Creatine prodrugs, compositions and methods of use thereof

ABSTRACT

The invention describes membrane permeable creatine prodrugs, pharmaceutical compositions comprising membrane permeable creatine prodrugs, and methods of treating diseases such as ischemia, heart failure, neurodegenerative disorders and genetic disorders affecting the creatine kinase system comprising administering creatine prodrugs or pharmaceutical compositions thereof. The invention also describes treating a genetic disease affecting the creatine kinase system, such as, for example, a creatine transporter disorder or a creatine synthesis disorder comprising administering creatine prodrugs or pharmaceutical compositions thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/481,568 filed on Apr. 7, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/975,950 filed on Dec. 21, 2015, now U.S. Pat.No. 9,617,230, which claims the benefit of U.S. Provisional ApplicationNo. 62/095,295, filed on Dec. 22, 2014 and entitled “CREATINE PRODRUGS,COMPOSITIONS AND METHODS OF USE THEREOF”, the disclosures of which arehereby incorporated by reference in their entireties for all purposes.

FIELD OF THE INVENTION

The invention describes membrane permeable creatine prodrugs,pharmaceutical compositions comprising membrane permeable creatineprodrugs, and methods of treating diseases, such as, for example,ischemia, heart failure, neurodegenerative disorders and geneticdisorders affecting the creatine kinase system comprising administeringcreatine prodrugs or pharmaceutical compositions thereof. In someembodiments the invention describes treating a genetic disease affectingthe creatine kinase system, such as, for example, a creatine transporterdisorder or a creatine synthesis disorder comprising administeringcreatine prodrugs or pharmaceutical compositions thereof.

BACKGROUND OF THE INVENTION

Creatine plays an important part in cellular energy metabolism,constituting as high-energy phosphocreatine a significant muscularenergy reserve in addition to adenosine triphosphate (ATP). In theresting state of the muscle, ATP can transfer a phosphate group ontocreatine, so forming phosphocreatine, which is then in directequilibrium with ATP. During muscular work, it is of vital importance toreplenish ATP stores as rapidly as possible. Phosphocreatine isavailable for this purpose during the first seconds of maximum muscleload; this substance is capable in a very rapid reaction of transferringa phosphate group onto adenosine diphosphate by the enzyme creatinekinase, so reforming ATP. The creatine kinase system has a dual role inintracellular energy metabolism-functioning as an energy buffer torestore depleted ATP levels at sites of high ATP hydrolysis, and totransferring energy in the form of phosphocreatine from the mitochondriato other parts of the cell by a process involving intermediate energycarriers, several enzymatic reactions, and diffusion through variousintracellular structures.

Many pathological disease states arise from a dysfunction in energymetabolism. Cellular depletion of ATP stores, as occurs for exampleduring tissue ischemia, results in impaired tissue functions and celldeath. Of foremost medical relevance, ischemia-related cardiovasculardisease such as stroke and heart attack remains a leading cause of deathand morbidity in North America and Europe. Thus, strategies that canprevent or reverse ischemia-related tissue damage are expected to have amajor impact on public health. Energy depletion also contributes totissue damage during surgery and is a common cause of organ transplantfailure. Furthermore, reperfusion with oxygen-containing solutions canfurther exacerbate tissue health through production of oxygen radicals.Therefore, a method to rapidly restore ATP levels without causingreperfusion injury is likely to have many therapeutic applications.Neurodegenerative diseases such as Parkinson's disease, Alzheimer'sdisease, and Huntington's disease are associated with impaired energymetabolism, and strategies for improving ATP metabolism couldpotentially minimize loss of neurons and thereby improve the prognosisof patients with these diseases. Finally, impaired energy metabolism isan important factor in muscle fatigue and limits physical endurance.Therefore, a method of preventing or reversing ATP depletion in ischemicor metabolically active tissues is likely to have broad clinical utilityin a wide range of indications.

Creatine supplementation increases intracellular creatine phosphatelevels (Harris et al., Clinical Sci 1992, 83, 367-74). Creatine readilycrosses the blood-brain barrier in healthy individuals and braincreatine levels can be increased via oral administration (Dechent etal., Am J Physiol 1999, 277, R698-704). Prolonged creatinesupplementation can elevate the cellular pools of creatine phosphate andincrease resistance to tissue ischemia and muscle fatigue. Thus,although administration of creatine may have some therapeuticusefulness, a modified creatine molecule that is more stable and is morepermeable to barrier tissues and cellular membranes would have enhancedtherapeutic value.

Creatine prodrugs of the invention are designed to be stable inbiological fluids, to enter cells by either passive diffusion or activetransport, and to release creatine into the cellular cytoplasm. Suchprodrugs can also cross important barrier tissues such as the intestinalmucosa, the blood-brain barrier, and the blood-placental barrier.Because of the ability to pass through biological membranes, creatineprodrugs can restore and maintain energy homeostasis in ATP depletedcells via the creatine kinase system, and rapidly restore ATP levels toprotect tissues from further ischemic stress. Creatine prodrugs having ahigher free energy or lower affinity for creatine kinase, and which canregenerate ATP under more severe conditions of energy depletion are alsodisclosed. Creatine prodrugs of the invention can also be used todeliver sustained systemic concentrations of creatine. The invention isdirected to these, as well as other, important ends.

SUMMARY OF THE INVENTION

The present invention relates to membrane permeable creatine prodrugs,pharmaceutical compositions comprising the membrane permeable creatineprodrugs, and methods of using membrane permeable creatine prodrugs, andpharmaceutical compositions thereof. In some embodiments the inventiondescribes treating a genetic disease affecting the creatine kinasesystem, such as, for example, a creatine transporter disorder or acreatine synthesis disorder comprising administering creatine prodrugsor pharmaceutical compositions thereof.

In one embodiment, the invention describes compounds of Formula (I) or apharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof:

wherein the compound of Formula (I) is

I

wherein:

R is —CH₃ or —CD₃;

R¹ is hydrogen, —OR², —C(O)OR², —C(O)R²,

n is an integer from 1 to 2;

each R² is independently hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl,C₁₋₁₂ heteroalkyl, substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl,substituted C₃₋₁₂ cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀cycloalkylalkyl, C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀heterocycloalkylalkyl, C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂heteroaryl, substituted C₅₋₁₂ heteroaryl, C₆₋₂₀ arylalkyl, substitutedC₆₋₂₀ arylalkyl, C₆₋₂₀ heteroarylalkyl or substituted C₆₋₂₀heteroarylalkyl;

each R³ and R⁴ is independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl;

R²³ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₅₋₁₂ cycloalkyl,substituted C₅₋₁₂ cycloalkyl, C₅₋₁₂ aryl, and C₅₋₁₂ substituted aryl,—C(O)—OR²² or —C(O)—R²²;

R²² is C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl; and

R⁴⁸ is C₁₋₁₂ alkyl or substituted C₁₋₁₂ alkyl.

Yet another embodiment describes compounds of Formula (III) or apharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof:

wherein the compound of Formula (III) is:

wherein:

W is —CH₂OH or —C(O)OR;

R is —CH₃ or —CD₃;

R⁷ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl, substituted C₆₋₂₀ heteroarylalkyl, —C(O)R⁵, —C(O)OR⁵,—C(O)(NR³R⁴), —C(R³R⁴)—C(O)OR²², —C(R³R⁴)—(O)C(O) R²²,—C(R³R⁴)—(O)C(O)—OR²²,

n is an integer from 1 to 2;

each R³ and R⁴ is independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl; and

R⁵ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl;

R²³ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₅₋₁₂ cycloalkyl,substituted C₅₋₁₂ cycloalkyl, C₅₋₁₂ aryl, and C₅₋₁₂ substituted aryl,—C(O)—OR²² or —C(O)—R²²; and

R²² is C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl.

Yet another embodiment describes compounds of Formula (VI) or apharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof:

wherein the compound of Formula (VI) is:

wherein:

R is —CH₃ or —CD₃;

R¹⁰ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂heteroalkyl, substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl,substituted C₃₋₁₂ cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀cycloalkylalkyl, C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀heterocycloalkylalkyl, C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂heteroaryl, substituted C₅₋₁₂ heteroaryl, C₆₋₂₀ arylalkyl, substitutedC₆₋₂₀ arylalkyl, C₆₋₂₀ heteroarylalkyl, substituted C₆₋₂₀heteroarylalkyl, —C(O)R⁵, —C(O)OR⁵, —C(O)(NR³R⁴), —C(R³R⁴)—C(O)OR²²,—C(R³R⁴)—(O)C(O) R²², —C(R³R⁴)—(O)C(O)—OR²²;

R¹¹ and R¹² are each independently hydrogen or —OR¹³; or R¹¹ and R¹² areeach —C(O)R⁵, with the proviso that both R¹¹ and R¹² cannot be hydrogen;

R¹³ is independently hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl,C₁₋₁₂ heteroalkyl, substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl,substituted C₃₋₁₂ cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀cycloalkylalkyl, C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀heterocycloalkylalkyl, C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂heteroaryl, substituted C₅₋₁₂ heteroaryl, C₆₋₂₀ arylalkyl, substitutedC₆₋₂₀ arylalkyl, C₆₋₂₀ heteroarylalkyl, substituted C₆₋₂₀heteroarylalkyl —CH(OR⁵), —C(O)R⁵, —C(O)OR⁵ or —C(O)(NR³R⁴);

each R³ and R⁴ is independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl;

R⁵ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl;

R²³ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₅₋₁₂ cycloalkyl,substituted C₅₋₁₂ cycloalkyl, C₅₋₁₂ aryl, and C₅₋₁₂ substituted aryl,—C(O)—OR²² or —C(O)—R²²;

R²² is C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl; and

n is an integer from 1 to 3.

One other embodiment describes compounds of Formula (VII) or apharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof:

wherein the compound of Formula (VII) is:

wherein:

R is —CH₃ or —CD₃;

each R¹⁴ is independently hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂alkyl, C₁₋₁₂ heteroalkyl, substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂cycloalkyl, substituted C₃₋₁₂ cycloalkyl, C₄₋₂₀ cycloalkylalkyl,substituted C₄₋₂₀ cycloalkylalkyl, C₄₋₂₀ heterocycloalkylalkyl,substituted C₄₋₂₀ heterocycloalkylalkyl, C₅₋₁₂ aryl, substituted C₅₋₁₂aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂ heteroaryl, C₆₋₂₀ arylalkyl,substituted C₆₋₂₀ arylalkyl, C₆₋₂₀ heteroarylalkyl, substituted C₆₋₂₀heteroarylalkyl —CH(OR⁵), —C(O)R⁵, —C(O)OR⁵ or —C(O)(NR³R⁴);

each R³ and R⁴ is independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl; and

R⁵ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl.

In certain embodiments, the compounds of Formulae (I), (III), (VI), and(VII), can include the following features:

Each R is independently —CH₃.

Each R is independently —CD₃.

Each n is independently the integer 1.

Each n is independently the integer 2.

Each R², R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹⁴, R¹⁵, R¹⁸ and R²² is independentlyC₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₃₋₇ cycloalkyl, substituted C₃₋₇cycloalkyl, C₅₋₇ aryl or substituted C₅₋₇ aryl.

Each R², R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹⁴, R¹⁵, R¹⁸ and R²² is independentlyhydrogen, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl,dodecyl, 1,1-dimethoxyethyl, 1,1-diethoxyethyl, phenyl, 4-methoxyphenyl,benzyl, phenethyl, styryl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, 2-pyridyl, 3-pyridyl or 4-pyridyl.

Each R², R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹⁴, R¹⁵, R¹⁸ and R²² is independentlyhydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl,dodecyl, 1,1-diethoxyethyl, phenyl, cyclohexyl or 3-pyridyl.

Each R², R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹⁴, R¹⁵, R¹⁸ and R²² is independentlyhydrogen, methyl, ethyl, n-propyl, isopropyl, dodecyl, tert-butyl,phenyl or cyclohexyl.

Each R², R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹⁴, R¹⁵, R¹⁸ and R²² is independentlyethyl, isopropyl or dodecyl.

Each R³ and R⁴ is independently hydrogen.

Each R²³ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl.

Each R²³ is methyl.

Each substituent group is independently halogen, —NO₂, —OH, —NH₂, —CN,—CF₃, —OCF₃, ═O, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy orsubstituted C₁₋₁₂ alkoxy, —COOR^(10′) wherein R^(10′) is hydrogen, C₁₋₃alkyl or —(NR^(11′))₂ wherein each R^(11′) is independently hydrogen orC₁₋₃ alkyl.

In one embodiment, the compound of Formula (I) is a compound of Formula(X), Formula (XI), Formula (XII), Formula (XIII), Formula (XIV), Formula(XV), Formula (XVa) or Formula (XVb) or a pharmaceutically acceptablesalt, solvate, tautomer or stereoisomer thereof; wherein the compound ofFormula (X) is:

wherein R is —CH₃ or —CD₃;

wherein the compound of Formula (XI) is:

wherein R is —CH₃ or —CD₃; and

R²⁴ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl;

wherein the compound of Formula (XII) is:

wherein R is —CH₃ or —CD₃; and

R²⁵ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl;

wherein the compound of Formula (XIII) is:

wherein R is —CH₃ or —CD₃; and

R²⁶ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl;

wherein the compound of Formula (XIV) is:

wherein the compound of Formula (XV) is:

wherein R is —CH₃ or —CD₃;

wherein the compound of Formula (XVa) is:

wherein R is —CH₃ or —CD₃;

R³⁹ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl; and

R³ and R⁴ are each independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl;

wherein the compound of Formula (XVb) is:

wherein R is —CH₃ or —CD₃;

R³ and R⁴ are each independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl; and

R⁵³ is C₁₋₁₂ alkyl or substituted C₁₋₁₂ alkyl.

In yet another embodiment, the compound of Formula (III) is a compoundof Formula (XVII), Formula (XVIII) or Formula (XIX) or apharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof;

wherein the compound of Formula (XVII) is:

wherein R is —CH₃ or —CD₃;

R²⁹ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl, -cyclohexyl, —CH₂—C(O)OR⁴³, —CH₂—(O)C(O)R⁴³,—CH₂—(O)C(O)OR⁴³ or

R³⁹ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl;

R⁴³ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl; and

R³ and R⁴ are each independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl; wherein the compound of Formula (XVIII) is:

wherein R is —CH₃ or —CD₃;

wherein the compound of Formula (XIX) is:

wherein R is —CH₃ or —CD₃.

In still another embodiment, the compound of Formula (VI) is a compoundof Formula (XXII), Formula (XXIII), Formula (XXIV), Formula (XXV),Formula (XXVI), Formula (XXVII) or Formula (XXVIII) or apharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof;

wherein the compound of formula (XXII) is:

wherein the compound of Formula (XXIII) is:

wherein the compound of Formula (XXIV) is:

wherein the compound of Formula (XXV) is:

wherein the compound of Formula (XXVI) is:

wherein the compound of Formula (XXVII) is:

wherein the compound of Formula (XXVIII) is:

wherein R is —CH₃ or —CD₃;

R^(a) is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, tert-butyl;

R³² is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl, cyclohexyl,

—CH₂—C(O)OR⁴³, —CH₂—(O)C(O)R⁴³, —CH₂—(O)C(O)OR⁴³ or

R³⁹ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl;

R³³ is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,tert-butyl, dodecyl, phenyl or cyclohexyl;

R⁴³ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl; and

R³ and R⁴ are each independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl.

In yet another embodiment, the compound of Formula (VII) is a compoundof Formula (XXIX) or a pharmaceutically acceptable salt, solvate,tautomer or stereoisomer thereof;

wherein the compound of Formula (XXIX) is:

wherein R is —CH₃ or —CD₃; and

each R³⁴ is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,tert-butyl, dodecyl, phenyl or cyclohexyl.

In one embodiment, the invention describes compounds having thefollowing structures:

or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof.

In another embodiment, the invention describes pharmaceuticalcompositions comprising a therapeutically effective amount of at leastone compound of Formulae (I), (III), (VI), and (VII), and any subgeneraor species thereof, or a pharmaceutically acceptable salt, solvate,tautomer or stereoisomer thereof, or a pharmaceutically acceptablesolvate of any of the foregoing, and a pharmaceutically acceptablevehicle. In one embodiment, the invention describes pharmaceuticalcompositions comprising a therapeutically effective amount of at leastone compound as disclosed herein, or a pharmaceutically acceptable salt,solvate, tautomer or stereoisomer thereof, or a pharmaceuticallyacceptable solvate of any of the foregoing, and a pharmaceuticallyacceptable vehicle.

In some embodiments, the pharmaceutical compositions can be formulatedin one or more sustained release oral dosage forms.

In one embodiment, the pharmaceutical composition comprises at least onecompound of the present invention in an amount effective for thetreatment of a disease in a patient wherein the disease is ischemia,oxidative stress, a neurodegenerative disease, ischemic reperfusioninjury, a cardiovascular disease, a genetic disease affecting thecreatine kinase system, multiple sclerosis, a psychotic disorder, andmuscle fatigue; an amount sufficient to effect energy homeostasis in atissue or an organ affected by a disease; an amount effective for theenhancement of muscle strength in a patient; an amount effective for theimprovement of the viability of a tissue or an organ; or an amounteffective for the improvement of the viability of cells. In anotherembodiment, the pharmaceutical composition comprises at least onecompound of the present invention in an amount effective for thetreatment of a genetic disease affecting the creatine kinase system. Insome embodiments, the pharmaceutical composition comprises at least onecompound of the present invention in an amount effective for thetreatment of a creatine transporter disorder. In one embodiment, thepharmaceutical composition comprises at least one compound of thepresent invention in an amount effective for the treatment of a creatinesynthesis disorder.

In one embodiment the invention describes methods for treating a diseasein a patient associated with a dysfunction in energy metabolism such asischemia, oxidative stress, a neurodegenerative disease, includingamyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson'sdisease or Alzheimer's disease, ischemic reperfusion injury, acardiovascular disease, multiple sclerosis (MS), a psychotic disorder, agenetic disease affecting the creatine kinase system or muscle fatiguein a patient comprising administering to a patient in need of suchtreatment a therapeutically effective amount of at least one compound ofFormulae (I), (III), (VI), (VII), and any subgenera or species thereof,or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof, or a pharmaceutical composition comprising at least onecompound of Formulae (I), (III), (VI), (VII), and any subgenera orspecies thereof, or a pharmaceutically acceptable salt, solvate,tautomer or stereoisomer thereof.

In another embodiment, methods are described for treating a geneticdisease affecting the creatine kinase system, such as, for example, acreatine transporter disorder or a creatine synthesis disorder in apatient comprising administering to a patient in need of such treatmenta therapeutically effective amount of at least one compound of Formulae(I), (III), (VI), (VII), and any subgenera or species thereof, or apharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof, or a pharmaceutical composition comprising at least onecompound of Formulae (I), (III), (VI), (VII), and any subgenera orspecies thereof, or a pharmaceutically acceptable salt, solvate,tautomer or stereoisomer thereof.

In further embodiments, methods are described for enhancing musclestrength in a patient comprising administering to a patient in need ofsuch enhancement a therapeutically effective amount of at least onecompound of Formulae (I), (III), (VI), (VII), and any subgenera orspecies thereof, or a pharmaceutically acceptable salt, solvate,tautomer or stereoisomer thereof, or a pharmaceutical compositioncomprising at least one compound of Formulae (I), (III), (VI), (VII),and any subgenera or species thereof, or a pharmaceutically acceptablesalt, solvate, tautomer or stereoisomer thereof.

In yet one more embodiment, methods are described for increasing theviability of a tissue or an organ comprising contacting the tissue orthe organ with an effective amount of at least one compound of Formulae(I), (III), (VI), (VII), and any subgenera or species thereof, or apharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof, or a pharmaceutical composition comprising at least onecompound of Formulae (I), (III), (VI), (VII), and any subgenera orspecies thereof, or a pharmaceutically acceptable salt, solvate,tautomer or stereoisomer thereof.

In still one more embodiment, methods are described for improving theviability of isolated cells comprising contacting the cells with aneffective amount of at least one compound of Formulae (I), (III), (VI),(VII), and any subgenera or species thereof, or a pharmaceuticallyacceptable salt, solvate, tautomer or stereoisomer thereof, or apharmaceutical composition comprising at least one compound of Formulae(I), (III), (VI), (VII), and any subgenera or species thereof, or apharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof.

In another embodiment, methods are described for treating a diseaseassociated with oxidative stress are provided comprising administeringto a patient in need of such treatment an effective amount of at leastone compound of Formulae (I), (III), (VI), (VII), and any subgenera orspecies thereof, or a pharmaceutically acceptable salt, solvate,tautomer or stereoisomer thereof, or a pharmaceutical compositioncomprising at least one compound of Formulae (I), (III), (VI), (VII),and any subgenera or species thereof, or a pharmaceutically acceptablesalt, solvate, tautomer or stereoisomer thereof.

In one more embodiment, methods for improving the viability of a tissueor an organ are described for treating a tissue or organ manifesting adysfunction in energy metabolism are provided comprising contacting atleast one compound of Formulae (I), (III), (VI), (VII), and anysubgenera or species thereof, or a pharmaceutically acceptable salt,solvate, tautomer or stereoisomer thereof, or a pharmaceuticalcomposition comprising at least one compound of Formulae (I), (III),(VI), (VII), and any subgenera or species thereof, or a pharmaceuticallyacceptable salt, solvate, tautomer or stereoisomer thereof, with thetissue or organ.

In yet one more embodiment, methods are described for effecting energyhomeostasis in a tissue or an organ are provided comprising contactingat least one compound of Formulae (I), (III), (VI), (VII), and anysubgenera or species thereof, or a pharmaceutically acceptable salt,solvate, tautomer or stereoisomer thereof, or a pharmaceuticalcomposition comprising at least one compound of Formulae (I), (III),(VI), (VII), and any subgenera or species thereof, or a pharmaceuticallyacceptable salt, solvate, tautomer or stereoisomer thereof, with thetissue or the organ.

In another embodiment, methods are described for treating an oxidativelystressed tissue or organ are provided comprising contacting at least onecompound of Formulae (I), (III), (VI), (VII), and any subgenera orspecies thereof, or a pharmaceutically acceptable salt, solvate,tautomer or stereoisomer thereof, or a pharmaceutical compositioncomprising at least one compound of Formulae (I), (III), (VI), (VII),and any subgenera or species thereof, or a pharmaceutically acceptablesalt, solvate, tautomer or stereoisomer thereof, with the tissue ororgan.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION Definitions

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a moiety or substituent. For example,—CONH2 is attached through the carbon atom.

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated, branched or straight-chain, monovalenthydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane, alkene or alkyne. Examples ofalkyl groups include, but are not limited to, methyl; ethyls such asethanyl, ethenyl, and ethynyl; propyls such as propan-1-yl, propan-2-yl,prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-1-yn-1-yl,prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl, 2-methyl-propan-2-yl, but-1-en-1-yl,but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, but-1-yn-1-yl, but-1-yn-3-yl,but-3-yn-1-yl, etc.; and the like.

The term “alkyl” is specifically intended to include groups having anydegree or level of saturation, i.e., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon-carbonbonds, groups having one or more triple carbon-carbon bonds, and groupshaving mixtures of single, double, and triple carbon-carbon bonds. Wherea specific level of saturation is intended, the terms “alkanyl,”“alkenyl,” and “alkynyl” are used. In certain embodiments, an alkylgroup can have from 1 to 20 carbon atoms, in certain embodiments, from 1to 12 carbon atoms, in certain embodiments, from 1 to 10 carbon atoms,in certain embodiments, from 1 to 6 carbon atoms, and in certainembodiments, from 1 to 3 carbon atoms.

“Alkoxy” by itself or as part of another substituent refers to a radicalOR³¹ where R³¹ is chosen from alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl,heteroaryl, arylalkyl, and heteroarylalkyl, as defined herein. Examplesof alkoxy groups include, but are not limited to, methoxy, ethoxy,propoxy, butoxy, cyclohexyloxy, and the like.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of a parent aromatic ringsystem. Aryl encompasses 5- and 6-membered carbocyclic aromatic rings,for example, benzene; bicyclic ring systems wherein at least one ring iscarbocyclic and aromatic, for example, naphthalene, indane, andtetralin; and tricyclic ring systems wherein at least one ring iscarbocyclic and aromatic, for example, fluorene. Aryl encompassesmultiple ring systems having at least one carbocyclic aromatic ringfused to at least one carbocyclic aromatic ring, cycloalkyl ring orheterocycloalkyl ring. For example, aryl includes 5- and 6-memberedcarbocyclic aromatic rings fused to a 5- to 7-membered heterocycloalkylring containing one or more heteroatoms chosen from N, O, and S. Forsuch fused, bicyclic ring systems wherein only one of the rings is acarbocyclic aromatic ring, the point of attachment may be at thecarbocyclic aromatic ring or the heterocycloalkyl ring. Examples of arylgroups include, but are not limited to, groups derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexylene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene, and the like. In certain embodiments, an aryl group canhave from 6 to 20 carbon atoms, from 6 to 12 carbon atoms, and incertain embodiments, from 6 to 8 carbon atoms. Aryl, however, does notencompass or overlap in any way with heteroaryl, separately definedherein.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Examples of arylalkyl groups include, but are not limitedto, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylalkenyl or arylalkynylis used. In certain embodiments, an arylalkyl group is C₆₋₃₀ arylalkyl,e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group isC.sub.1-10 and the aryl moiety is C₆₋₂₀, in certain embodiments, anarylalkyl group is C₆₋₂₀arylalkyl, e.g., the alkanyl, alkenyl or alkynylmoiety of the arylalkyl group is C₁₋₈ and the aryl moiety is C₆₋₁₂.

“AUC” is the area under a curve representing the concentration of acompound or metabolite thereof in a biological fluid in a patient as afunction of time following administration of the compound to thepatient. In certain embodiments, the compound can be a prodrug and themetabolite can be a drug. Examples of biological fluids include plasmaand blood. The AUC may be determined by measuring the concentration of acompound or metabolite thereof in a biological fluid such as the plasmaor blood using methods such as liquid chromatography-tandem massspectrometry (LC/MS/MS), at various time intervals, and calculating thearea under the plasma concentration-versus-time curve. Suitable methodsfor calculating the AUC from a drug concentration-versus-time curve arewell known in the art. As relevant to the invention, an AUC for a drugor metabolite thereof may be determined by measuring over time theconcentration of the drug in the plasma, blood or other biological fluidor tissue of a patient following administration of a correspondingcompound of the invention to the patient.

“Bioavailability” refers to the rate and amount of a drug that reachesthe systemic circulation of a patient following administration of thedrug or prodrug thereof to the patient and can be determined byevaluating, for example, the plasma or blood concentration-versus-timeprofile for a drug. Parameters useful in characterizing a plasma orblood concentration-versus-time curve include the area under the curve(AUC), the time to maximum concentration (T_(max)), and the maximum drugconcentration (C_(max)), where C_(max) is the maximum concentration of adrug in the plasma or blood of a patient following administration of adose of the drug or form of drug to the patient, and T_(max) is the timeto the maximum concentration (C_(max)) of a drug in the plasma or bloodof a patient following administration of a dose of the drug or form ofdrug to the patient.

“C_(max)” is the maximum concentration of a drug in the plasma or bloodof a patient following administration of a dose of the drug or prodrugto the patient.

“T_(max)” is the time to the maximum (peak) concentration (C_(max)) of adrug in the plasma or blood of a patient following administration of adose of the drug or prodrug to the patient.

“Compounds of the invention” or “compound of the invention”, include anyspecific compounds within these formulae. Compounds may be identifiedeither by their chemical structure and/or chemical name. When thechemical structure and chemical name are conflicting, the chemicalstructure is determinative of the identity of the compound. Thecompounds described herein may comprise one or more chiral centersand/or double bonds and therefore may exist as stereoisomers such asdouble-bond isomers (i.e., geometric isomers), enantiomers ordiastereomers. Accordingly, any chemical structures within the scope ofthe specification depicted, in whole or in part, with a relativeconfiguration encompass all possible enantiomers and stereoisomers ofthe illustrated compounds including the stereoisomerically pure form(e.g., geometrically pure, enantiomerically pure or diastereomericallypure) and enantiomeric and stereoisomeric mixtures. Enantiomeric andstereoisomeric mixtures may be resolved into their component enantiomersor stereoisomers using separation techniques or chiral synthesistechniques well known to the skilled artisan. Compounds of the inventionare also referred to as “prodrugs of creatine” or “prodrugs of theinvention.”

Compounds of invention include, but are not limited to, stereoisomers oroptical isomers of compounds of the invention, racemates thereof, andother mixtures thereof. In such embodiments, the single enantiomers ordiastereomers, i.e., optically active forms, can be obtained byasymmetric synthesis or by resolution of the racemates. Resolution ofthe racemates may be accomplished, for example, by conventional methodssuch as crystallization in the presence of a resolving agent orchromatography, using, for example a chiral high-pressure liquidchromatography (HPLC) column. In addition, compounds of the inventioninclude Z- and E-forms (or cis- and trans-forms) of compounds withdouble bonds. In embodiments in which compounds of the invention existin various tautomeric forms, the compounds include all tautomeric formsof the compound.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposablemirror images of one another.

Compounds of the invention may also exist in several tautomeric forms,and the depiction herein of one tautomer is for convenience only, and isalso understood to encompass other tautomers of the form shown.Accordingly, the chemical structures depicted herein encompass allpossible tautomeric forms of the illustrated compounds. The term“tautomer” as used herein refers to isomers that change into one anotherwith great ease so that they can exist together in equilibrium. Forexample, ketone and enol are two tautomeric forms of one compound. Inanother example, a substituted 1,2,4-triazole derivative may exist in atleast three tautomeric forms as shown below:

Compounds of the invention also include isotopically labeled compoundswhere one or more atoms have an atomic mass different from the atomicmass conventionally found in nature. Examples of isotopes that may beincorporated into the compounds disclosed herein include, but are notlimited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, etc. Compounds mayexist in unsolvated forms as well as solvated forms, including hydratedforms and as N-oxides. In general, compounds may be hydrated, solvatedor N-oxides. Certain compounds may exist in multiple crystalline oramorphous forms. Compounds of the invention include pharmaceuticallyacceptable salts thereof or pharmaceutically acceptable solvates of thefree acid form of any of the foregoing, as well as crystalline forms ofany of the foregoing.

“Creatine kinase system” includes, but is not limited to the creatinetransporter, creatine, creatine kinase, creatine phosphate, and theintracellular energy transport of creatine, creatine kinase, and/orcreatine phosphate. The creatine kinase system includes mitochondrialand cytoplasmic creatine kinase systems. Affecting the creatine kinasesystem refers to the transport, synthesis, metabolism, translocation,and the like, of the compounds and proteins comprising the creatinekinase system.

“Cycloalkyl” by itself or as part of another substituent refers to asaturated or partially unsaturated cyclic alkyl radical. Where aspecific level of saturation is intended, the nomenclature“cycloalkanyl” or “cycloalkenyl” is used. Examples of cycloalkyl groupsinclude, but are not limited to, groups derived from cyclopropane,cyclobutane, cyclopentane, cyclohexane, and the like. In certainembodiments, a cycloalkyl group is C₃₋₁₅ cycloalkyl, C₅₋₁₂ cycloalkyl,and in certain embodiments, C₃₋₇ cycloalkyl.

“Cycloalkylalkyl” by itself or as part of another substituent refers toan acyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with acycloalkyl group. Where specific alkyl moieties are intended, thenomenclature cycloalkylalkanyl, cycloalkylalkenyl or cycloalkylalkynylis used. In certain embodiments, a cycloalkylalkyl group is C₇₋₃₀cycloalkylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thecycloalkylalkyl group is C₁₋₁₀ and the cycloalkyl moiety is C₆₋₂₀, andin certain embodiments, a cycloalkylalkyl group is C₇₋₂₀cycloalkylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thecycloalkylalkyl group is C₁₋₈ and the cycloalkyl moiety is C₄₋₂₀ orC₆₋₁₂.

“Disease” refers to a disease, disorder, condition, symptom orindication.

“Halogen” refers to a fluoro, chloro, bromo or iodo group.

“Heteroalkyl” by itself or as part of another substituent refer to analkyl group in which one or more of the carbon atoms (and any associatedhydrogen atoms) are each independently replaced with the same ordifferent heteroatomic groups. Examples of heteroatomic groups include,but are not limited to, —O—, —S—, —O—O—, —S—S—, —O—S—, —NR⁵⁷R⁵⁸—, ═N—N═,—N═N—, —N═N—NR⁵⁹R⁶⁰, —PR⁶¹—, —P(O)₂—, —POR⁶²—, —O—P(O)₂—, —SO—, —SO₂—,—SnR⁶³R⁶⁴—, and the like, where R⁵⁷, R⁵, R⁵⁹, R⁶⁰, R⁶¹, R⁶², R⁶³, andR⁶⁴ are each independently chosen from hydrogen, C₁₋₁₂ alkyl,substituted C₁₋₁₂ alkyl, C₆₋₁₂ aryl, substituted C₆₋₁₂ aryl, C₇₋₈arylalkyl, substituted C₇₋₈ arylalkyl, C₃₋₇ cycloalkyl, substituted C₃₋₇cycloalkyl, C₃₋₇ heterocycloalkyl, substituted C₃₋₇ heterocycloalkyl,C₁₋₁₂ heteroalkyl, substituted C₁₋₁₂ heteroalkyl, C₆₋₁₂ heteroaryl,substituted C₆₋₁₂ heteroaryl, C₇₋₁₈ heteroarylalkyl or substituted C₇₋₈heteroarylalkyl. Where a specific level of saturation is intended, thenomenclature “heteroalkanyl,” “heteroalkenyl,” or R⁶⁰, R⁶¹, R⁶², R⁶³,and R⁶⁴ “heteroalkynyl” is used. In certain embodiments, R⁵⁷, R⁵⁸, R⁵⁹,are each independently chosen from hydrogen and C₁₋₃ alkyl.

“Heteroaryl” by itself or as part of another substituent refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a parent heteroaromatic ring system.Heteroaryl encompasses multiple ring systems having at least oneheteroaromatic ring fused to at least one other ring, which can bearomatic or non-aromatic. Heteroaryl encompasses 5- to 7-memberedaromatic, monocyclic rings containing one or more, for example, from 1to 4 or in certain embodiments, from 1 to 3, heteroatoms chosen from N,O, and S, with the remaining ring atoms being carbon; and bicyclicheterocycloalkyl rings containing one or more, for example, from 1 to 4or in certain embodiments, from 1 to 3, heteroatoms chosen from N, O,and S, with the remaining ring atoms being carbon and wherein at leastone heteroatom is present in an aromatic ring. For example, heteroarylincludes a 5- to 7-membered heteroaromatic ring fused to a 5- to7-membered cycloalkyl ring. For such fused, bicyclic heteroaryl ringsystems wherein only one of the rings contains one or more heteroatoms,the point of attachment may be at the heteroaromatic ring or thecycloalkyl ring. In certain embodiments, when the total number of N, S,and O atoms in the heteroaryl group exceeds one, the heteroatoms are notadjacent to one another. In certain embodiments, the total number of N,S, and O atoms in the heteroaryl group is not more than two. In certainembodiments, the total number of N, S, and O atoms in the aromaticheterocycle is not more than one. Heteroaryl does not encompass oroverlap with aryl as defined herein.

Examples of heteroaryl groups include, but are not limited to, groupsderived from acridine, arsindole, carbazole, β-carboline, chromane,chromene, cinnoline, furan, imidazole, indazole, indole, indoline,indolizine, isobenzofuran, isochromene, isoindole, isoindoline,isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole,oxazole, perimidine, phenanthridine, phenanthroline, phenazine,phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine,pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline,quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene,triazole, xanthene, and the like. In certain embodiments, a heteroarylgroup is from 5- to 20-membered heteroaryl, in certain embodiments from5- to 10-membered heteroaryl, and in certain embodiments from 6- to8-heteroaryl. In certain embodiments heteroaryl groups are those derivedfrom thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine,quinoline, imidazole, oxazole or pyrazine.

“Heteroarylalkyl” by itself or as part of another substituent refers toan acyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, is replaced with a heteroaryl group. Typically a terminalor sp³ carbon atom is the atom replaced with the heteroaryl group. Wherespecific alkyl moieties are intended, the nomenclature“heteroarylalkanyl,” “heteroarylalkenyl,” and “heterorylalkynyl” isused. In certain embodiments, a heteroarylalkyl group is a 6- to30-membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynylmoiety of the heteroarylalkyl is 1- to 10-membered and the heteroarylmoiety is a 5- to 20-membered heteroaryl, and in certain embodiments, 6-to 20-membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynylmoiety of the heteroarylalkyl is 1- to 8-membered and the heteroarylmoiety is a 5- to 12-membered heteroaryl.

“Heterocycloalkyl” by itself or as part of another substituent refers toa partially saturated or unsaturated cyclic alkyl radical in which oneor more carbon atoms (and any associated hydrogen atoms) are eachindependently replaced with the same or different heteroatom. Examplesof heteroatoms to replace the carbon atom(s) include, but are notlimited to, N, P, O, S, Si, etc. Where a specific level of saturation isintended, the nomenclature “heterocycloalkanyl” or “heterocycloalkenyl”is used. Examples of heterocycloalkyl groups include, but are notlimited to, groups derived from epoxides, azirines, thiiranes,imidazolidine, morpholine, piperazine, piperidine, pyrazolidine,pyrrolidine, quinuclidine, and the like.

“Heterocycloalkylalkyl” by itself or as part of another substituentrefers to an acyclic alkyl radical in which one of the hydrogen atomsbonded to a carbon atom, typically a terminal or sp³carbon atom, isreplaced with a heterocycloalkyl group. Where specific alkyl moietiesare intended, the nomenclature heterocycloalkylalkanyl,heterocycloalkylalkenyl or heterocycloalkylalkynyl is used. In certainembodiments, a heterocycloalkylalkyl group is a 6- to 30-memberedheterocycloalkylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety ofthe heterocycloalkylalkyl is 1- to 10-membered and the heterocycloalkylmoiety is a 5- to 20-membered heterocycloalkyl, and in certainembodiments, 6- to 20-membered heterocycloalkylalkyl, e.g., the alkanyl,alkenyl or alkynyl moiety of the heterocycloalkylalkyl is 1- to8-membered and the heterocycloalkyl moiety is a 5- to 12-memberedheterocycloalkyl.

“Leaving group” refers to an atom or a group capable of being displacedby a nucleophile and includes halogen, such as chloro, bromo, fluoro,and iodo, alkoxycarbonyl (e.g., acetoxy), aryloxycarbonyl, mesyloxy,tosyloxy, trifluoromethanesulfonyloxy, aryloxy (e.g.,2,4-dinitrophenoxy), methoxy, N,O-dimethylhydroxylamino, and the like.

“Parent aromatic ring system” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated n electron system. Includedwithin the definition of “parent aromatic ring system” are fused ringsystems in which one or more of the rings are aromatic and one or moreof the rings are saturated or unsaturated, such as, for example,fluorene, indane, indene, phenalene, etc. Examples of parent aromaticring systems include, but are not limited to, aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexylene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like.

“Parent heteroaromatic ring system” refers to an aromatic ring system inwhich one or more carbon atoms (and any associated hydrogen atoms) areeach independently replaced with the same or different heteroatom.Examples of heteroatoms to replace the carbon atoms include, but are notlimited to, N, P, O, S, and Si, etc. Specifically included within thedefinition of “parent heteroaromatic ring systems” are fused ringsystems in which one or more of the rings are aromatic and one or moreof the rings are saturated or unsaturated, such as, for example,arsindole, benzodioxan, benzofuran, chromane, chromene, indole,indoline, xanthene, etc. Examples of parent heteroaromatic ring systemsinclude, but are not limited to, arsindole, carbazole, β-carboline,chromane, chromene, cinnoline, furan, imidazole, indazole, indole,indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like.

“Patient” refers to animals, preferably mammals, most preferably humans,and includes males and females, and children and adults.

“Pharmaceutical composition” refers to at least one compound of theinvention and at least one pharmaceutically acceptable vehicle, withwhich the at least one compound of the invention is administered to apatient, contacted with a tissue or organ or contacted with a cell.“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals, and more particularly in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound, whichpossesses the desired pharmacological activity of the parent compound.Such salts include: (1) acid addition salts, formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; and (2)salts formed when an acidic proton present in the parent compound isreplaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine, andthe like. In certain embodiments, a pharmaceutically acceptable salt isthe hydrochloride salt.

“Pharmaceutically acceptable vehicle” refers to a pharmaceuticallyacceptable diluent, a pharmaceutically acceptable adjuvant, apharmaceutically acceptable excipient, a pharmaceutically acceptablecarrier or a combination of any of the foregoing with which a compoundof the invention may be administered to a patient and which does notdestroy the pharmacological activity thereof and which is non-toxic whenadministered in doses sufficient to provide a therapeutically effectiveamount of the compound.

“Prodrug” refers to a derivative of a drug molecule that requires atransformation within the body to release the active drug. Prodrugs arefrequently, although not necessarily, pharmacologically inactive untilconverted to the parent drug. Compounds of Formulae (I), (III), (VI),(VII), and any subgenera or species thereof, are prodrugs of creatinethat can be metabolized within a patient's body to release creatine.

“Promoiety” refers to a group bonded to a drug, typically to afunctional group of the drug, via bond(s) that are cleavable underspecified conditions of use. The bond(s) between the drug and promoietymay be cleaved by enzymatic or non-enzymatic means. Under the conditionsof use, for example following administration to a patient, the bond(s)between the drug and promoiety may be cleaved to release the parentdrug. The cleavage of the promoiety may proceed spontaneously, such asvia a hydrolysis reaction or it may be catalyzed or induced by anotheragent, such as by an enzyme, by light, by acid or by a change of orexposure to a physical or environmental parameter, such as a change oftemperature, pH, etc. The agent may be endogenous to the conditions ofuse, such as an enzyme present in the systemic circulation of a patientto which the prodrug is administered or the acidic conditions of thestomach or the agent may be supplied exogenously.

“Protecting group” refers to a grouping of atoms, which when attached toa reactive group in a molecule masks, reduces or prevents thatreactivity. Examples of protecting groups can be found in Wuts andGreene, “Protective Groups in Organic Synthesis,” John Wiley & Sons, 4thed. 2006; Harrison et al., “Compendium of Organic Synthetic Methods,”Vols. 1-11, John Wiley & Sons 1971-2003; Larock “Comprehensive OrganicTransformations,” John Wiley & Sons, 2nd ed. 2000; and Paquette,“Encyclopedia of Reagents for Organic Synthesis,” John Wiley & Sons,11th ed. 2003. Examples of amino protecting groups include, but are notlimited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl(CBZ), tert-butoxycarbonyl (Boc), trimethylsilyl (TMS),2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted tritylgroups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC),nitro-veratryloxycarbonyl (NVOC), and the like. Examples of hydroxyprotecting groups include, but are not limited to, those in which thehydroxy group is either acylated or alkylated such as benzyl, and tritylethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilylethers, and allyl ethers.

“Solvate” refers to a molecular complex of a compound with one or moresolvent molecules in a stoichiometric or non-stoichiometric amount. Suchsolvent molecules are those commonly used in the pharmaceutical art,which are known to be innocuous to recipient, e.g., water, ethanol, andthe like. A molecular complex of a compound or moiety of a compound anda solvent can be stabilized by non-covalent intra-molecular forces suchas, for example, electrostatic forces, van der Waals forces or hydrogenbonds. The term “hydrate” refers to a complex in which the one or moresolvent molecules are water including monohydrates and hemi-hydrates.

“Substantially one diastereomer” refers to a compound containing two ormore stereogenic centers such that the diastereomeric excess (d.e.) ofthe compound is greater than or about at least 90%. In certainembodiments, the d.e. is, for example, greater than or at least about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99%.

“Substituted” refers to a group in which one or more hydrogen atoms areeach independently replaced with the same or different substituent(s).Examples of substituents include, but are not limited to, -M, —R⁷⁰, —O⁻,═O, —OR⁷⁰, —SR.⁷⁰, —S⁻, ═S, —NR⁷⁰R⁷¹, ═NR⁷⁰, —CF₃, —CN, —OCN, —SCN, —NO,—NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R⁷⁰, —OS(O₂)O⁻, —OS(O)₂R⁷⁰,—P(O)(O⁻)₂, —P(O)(OR⁷⁰)(O⁻), —OP(O)(OR⁷⁰)(OR⁷¹), —C(O)R⁷⁰, —C(S)R⁷⁰,—C(O)OR⁷⁰, —C(O)NR⁷⁰R⁷¹, —C(O)O—, —C(S)OR⁷⁰, —NR⁷²C(O)NR⁷⁰R⁷¹,—NR⁷²C(S)NR⁷⁰R⁷¹, —NR⁷²C(NR⁷³)NR⁷⁰R⁷¹, and —C(NR⁷²)NR⁷⁰R⁷¹ where M isindependently a halogen; R⁷⁰, R⁷¹, R⁷², and R⁷³ are each independentlychosen from hydrogen, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl,and heteroaryl or R⁷⁰ and R⁷¹ together with the nitrogen atom to whichthey are bonded form a ring chosen from a heterocycloalkyl ring. Incertain embodiments, R⁷⁰, R⁷¹, R⁷², and R⁷³ are each independentlychosen from hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₃₋₁₂ cycloalkyl, C₃₋₁₂heterocycloalkyl, C₆₋₁₂ aryl, and C₆₋₁₂ heteroaryl. In certainembodiments, each substituent is independently selected from halogen,—OH, —CN, —CF₃, ═O, —NO₂, C₁₋₃ alkoxy, C₁₋₃ alkyl, —COOR⁸⁰ wherein R⁸⁰is selected from hydrogen, C₁₋₃ alkyl and (NR⁷⁴)₂ wherein each R⁷⁴ isindependently hydrogen or C₁₋₃ alkyl.

In certain embodiments, substituted aryl and substituted heteroarylinclude one or more of the following substitute groups: F, Cl, Br, C₁₋₃alkyl, substituted alkyl, C₁₋₃ alkoxy, —S(O)₂NR⁵⁰R⁵¹, —NR⁵⁰R⁵¹, —CF₃,—OCF₃, —CN, —NR⁵⁰S(O)₂R⁵¹, —NR⁵⁰C(O)R⁵¹, C₅₋₁₀ aryl, substituted C₅₋₁₀aryl, C₅₋₁₀ heteroaryl, substituted C₅₋₁₀ heteroaryl, —C(O)OR⁵⁰, —NO₂,—C(O)R⁵⁰, —C(O)NR⁵⁰R⁵¹, —OCHF₂, C₁₋₃ acyl, —SR⁵⁰, —S(O)₂OH, —S(O)₂R⁵⁰,—S(O)R⁵⁰, —C(S)R⁵⁰, —C(O)O—, —C(S)OR⁵⁰S, —NR⁵⁰C(O)NR⁵¹R⁵²,—NR⁵⁰C(S)NR⁵¹R⁵², and —C(NR⁵⁰)NR⁵¹R⁵², C₃₋₈ cycloalkyl, and substitutedC₃₋₈ cycloalkyl, wherein R⁵⁰, R⁵¹, and R⁵² are each independentlyselected from hydrogen and C₁₋₄ alkyl.

In certain embodiments, a substituent group can be selected fromhalogen, —NO₂, —OH, —COOH, —NH₂, —CN, —CF₃, —OCF₃, C₁₋₈ alkyl,substituted C₁₋₈ alkyl, C₁₋₈ alkoxy, and substituted C₁₋₈ alkoxy,wherein the each substituent of the substituted C₁₋₈ alkyl and C₁₋₈alkoxy is independently selected from halogen, —NO₂, —OH, —COOH, —NH₂,—CN, —CF₃, —OCF₃.

In certain embodiments, each substituent is independently selected fromhalogen, —OH, —CN, —CF₃, ═O, —NO₂, C₁₋₃ alkoxy, C₁₋₃ alkyl, —COOR⁸⁰wherein R⁸⁰ is selected from hydrogen, C₁₋₃ alkyl and (NR⁷⁴)₂ whereineach R⁷⁴ is independently hydrogen or C₁₋₃ alkyl.

“Therapeutically effective amount” refers to the amount of a compoundthat, when administered to a subject for treating a disease or disorderor at least one of the clinical symptoms of a disease or disorder, issufficient to affect such treatment of the disease, disorder or symptom.The “therapeutically effective amount” can vary depending, for example,on the compound, the disease, disorder, and/or symptoms of the diseaseor disorder, severity of the disease, disorder, and/or symptoms of thedisease or disorder, the age, weight, and/or health of the patient to betreated, and the judgment of the prescribing physician. An appropriateamount in any given instance can be readily ascertained by those skilledin the art or capable of determination by routine experimentation.

“Therapeutically effective dose” refers to a dose that provideseffective treatment of a disease or disorder in a patient. Atherapeutically effective dose may vary from compound to compound, andfrom patient to patient, and may depend upon factors such as thecondition of the patient and the route of delivery. A therapeuticallyeffective dose may be determined in accordance with routinepharmacological procedures known to those skilled in the art.

“Treating” or “treatment” of any disease or disorder refers to arrestingor ameliorating a disease, disorder or at least one of the clinicalsymptoms of a disease or disorder, reducing the risk of acquiring adisease, disorder or at least one of the clinical symptoms of a diseaseor disorder, reducing the development of a disease, disorder or at leastone of the clinical symptoms of the disease or disorder or reducing therisk of developing a disease or disorder or at least one of the clinicalsymptoms of a disease or disorder. “Treating” or “treatment” also refersto inhibiting the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter) or both, and to inhibiting atleast one physical parameter that may or may not be discernible to thepatient. In certain embodiments, “treating” or “treatment” refers todelaying the onset of the disease or disorder or at least one or moresymptoms thereof in a patient which may be exposed to or predisposed toa disease or disorder even though that patient does not yet experienceor display symptoms of the disease or disorder.

Reference is now made in detail to certain embodiments of compounds,compositions, and methods. The disclosed embodiments are not intended tobe limiting of the claims. To the contrary, the claims are intended tocover all alternatives, modifications, and equivalents.

Creatine Prodrugs

In certain embodiments, a creatine prodrug is a compound of Formula (I)or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof:

wherein the compound of Formula (I) is

wherein:

R is —CH₃ or —CD₃;

R¹ is hydrogen, —OR², —C(O)OR², —C(O)R²,

n is an integer from 1 to 2;

each R² is independently hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl,C₁₋₁₂ heteroalkyl, substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl,substituted C₃₋₁₂ cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀cycloalkylalkyl, C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀heterocycloalkylalkyl, C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂heteroaryl, substituted C₅₋₁₂ heteroaryl, C₆₋₂₀ arylalkyl, substitutedC₆₋₂₀ arylalkyl, C₆₋₂₀ heteroarylalkyl or substituted C₆₋₂₀heteroarylalkyl;

each R³ and R⁴ is independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl;

R²³ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₅₋₁₂ cycloalkyl,substituted C₅₋₁₂ cycloalkyl, C₅₋₁₂ aryl, and C₅₋₁₂ substituted aryl,—C(O)—OR²² or —C(O)—R²²;

R²² is C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl; and

R⁴⁸ is C₁₋₁₂ alkyl or substituted C₁₋₁₂ alkyl.

In certain embodiments of a compound of Formula (I), n is the integer 1.

In certain embodiments of a compound of Formula (I), n is the integer 2.

In certain embodiments of a compound of Formula (I) each R² and R²² isindependently hydrogen, methyl, ethyl, n-propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl,neopentyl, dodecyl, 1,1-dimethoxyethyl, 1,1-diethoxyethyl, phenyl,4-methoxyphenyl, benzyl, phenethyl, styryl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, 2-pyridyl, 3-pyridyl or 4-pyridyl.

In certain embodiments of a compound of Formula (I), each R² and R²² isindependently hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl,neopentyl, dodecyl, 1,1-diethoxyethyl, phenyl, cyclohexyl or 3-pyridyl.

In certain embodiments of a compound of Formula (I), each R² and R²² isindependently hydrogen, methyl, ethyl, n-propyl, isopropyl, dodecyl,tert-butyl, phenyl or cyclohexyl.

In certain embodiments of a compound of Formula (I), each R² and R²² isindependently ethyl, isopropyl or dodecyl.

In certain embodiments of a compound of Formula (I), R³ and R⁴ eachindependently is hydrogen.

In certain embodiments of a compound of Formula (I), R²³ is hydrogen,methyl, ethyl, n-propyl, isopropyl, tert-butyl, dodecyl, phenyl orcyclohexyl.

In certain embodiments of a compound of Formula (I), R²³ is methyl.

In certain embodiments of a compound of Formula (I), each substituentgroup is independently halogen, —NO₂, —OH, —NH₂, —CN, —CF₃, —OCF₃, ═O,C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy or substituted C₁₋₁₂alkoxy, —COOR^(10′) wherein R^(10′) is hydrogen, C₁₋₃ alkyl or—(NR^(11′))₂ wherein each R^(11′) is independently hydrogen or C₁₋₃alkyl.

In some embodiment, the compound of Formula (I) is a compound of Formula(X), Formula (XI), Formula (XII), Formula (XIII), Formula (XIV), Formula(XV), Formula (XVa) or Formula (XVb) or a pharmaceutically acceptablesalt, solvate, tautomer or stereoisomer thereof;

wherein the compound of Formula (X) is:

wherein R is —CH₃ or —CD₃;

wherein the compound of Formula (XI) is:

wherein R is —CH₃ or —CD₃; and

R²⁴ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl;

wherein the compound of Formula (XII) is:

wherein R is —CH₃ or —CD₃; and

R²⁵ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl;

wherein the compound of Formula (XIII) is:

wherein R is —CH₃ or —CD₃; and

R²⁶ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl;

wherein the compound of Formula (XIV) is:

wherein R is —CH₃ or —CD₃;

wherein the compound of Formula (XV) is:

wherein R is —CH₃ or —CD₃;

wherein the compound of Formula (XVa) is:

wherein R is —CH₃ or —CD₃;

R³⁹ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl, and

R³ and R⁴ is independently hydrogen, C₁₋₁₂ alkyl or substituted C₁₋₁₂alkyl;

wherein the compound of Formula (XVb) is:

wherein R is —CH₃ or —CD₃;

R³ and R⁴ are each independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl; and

R⁵³ is C₁₋₁₂ alkyl or substituted C₁₋₁₂ alkyl.

In certain embodiments in the compounds of Formula (XI), (XII) and(XIII), each R²⁴, R²⁵ and R²⁶ is independently ethyl, isopropyl ordodecyl.

In certain embodiments in the compounds of Formula (XVa), R³⁹ is methyl,ethyl, n-propyl, isopropyl, tert-butyl, dodecyl, phenyl or cyclohexyl.

In certain embodiments in the compounds of Formula (XVa), R³⁹ is methyl,

In certain embodiments in the compounds of Formula (XVa) or (XVb), R³and R⁴ are each hydrogen.

In certain embodiments in the compounds of Formula (XVb), R⁵³ is methyl,ethyl, n-propyl, isopropyl or tert-butyl.

In certain embodiments a creatine prodrug is a compound of Formula (III)or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof:

wherein the compound of Formula (III) is:

wherein:

W is —CH₂OH or —C(O)OR⁷;

R is —CH₃ or —CD₃;

R⁷ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl, substituted C₆₋₂₀ heteroarylalkyl, —C(O)R⁵, —C(O)OR⁵,—C(O)(NR³R⁴), —C(R³R⁴)—C(O)OR²², —C(R³R⁴)—(O)C(O) R²²,—C(R³R⁴)—(O)C(O)—OR²²,

n is an integer from 1 to 2;

each R³ and R⁴ is independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl; and

R⁵ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl;

R²³ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₅₋₁₂ cycloalkyl,substituted C₅₋₁₂ cycloalkyl, C₅₋₁₂ aryl, and C₅₋₁₂ substituted aryl,—C(O)—OR²² or —C(O)—R²²; and

R²² is C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl.

In certain embodiments of a compound of Formula (III), n is the integer1.

In certain embodiments of a compound of Formula (III), n is the integer2.

In certain embodiments of a compound of Formula (III), each R⁵, R⁷ andR²² is independently C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₃₋₇cycloalkyl, substituted C₃₋₇ cycloalkyl, C₅₋₇ aryl or substituted C₅₋₇aryl.

In certain embodiments of a compound of Formula (III), each R⁵, R⁷ andR²² is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl,neopentyl, dodecyl, 1,1-dimethoxyethyl, 1,1-diethoxyethyl, phenyl,4-methoxyphenyl, benzyl, phenethyl, styryl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, 2-pyridyl, 3-pyridyl or 4-pyridyl.

In certain embodiments of a compound of Formula (III), each R⁵, R⁷ andR²² is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl,sec-pentyl, neopentyl, dodecyl, 1,1-diethoxyethyl, phenyl, cyclohexyl or3-pyridyl.

In certain embodiments of a compound of Formula (III), each R⁵, R⁷ andR²² is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,dodecyl, tert-butyl, phenyl or cyclohexyl.

In certain embodiments of a compound of Formula (III), each R⁵, R⁷ andR²² is independently ethyl, isopropyl or dodecyl.

In certain embodiments of a compound of Formula (III), each R³ and R⁴ isindependently hydrogen.

In certain embodiments of a compound of Formula (III), each R²³ ishydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl, dodecyl,phenyl or cyclohexyl.

In certain embodiments of a compound of Formula (III), each R²³ ismethyl.

In certain embodiments of a compound of Formula (III), each substituentgroup is independently halogen, —NO₂, —OH, —NH₂, —CN, —CF₃, —OCF₃, ═O,C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy or substituted C₁₋₁₂alkoxy, —COOR^(10′) wherein R^(10′) is hydrogen, C₁₋₃ alkyl or—(NR^(11′))₂ wherein each R^(11′) is independently hydrogen or C₁₋₃alkyl.

In yet another embodiment, the compound of Formula (III) is a compoundof Formula (XVII), Formula (XVIII) or Formula (XIX) or apharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof;

wherein the compound of Formula (XVII) is:

wherein R is —CH₃ or —CD₃;

R²⁹ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl, -cyclohexyl, —CH₂—C(O)OR⁴³, —CH₂—(O)C(O)R⁴³,—CH₂—(O)C(O)OR⁴³ or

R³⁹ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl;

R⁴³ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl; and

R³ and R⁴ are each independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl;

wherein the compound of Formula (XVIII) is:

wherein R is —CH₃ or —CD₃;

wherein the compound of Formula (XIX) is:

wherein R is —CH₃ or —CD₃.

In certain embodiments of a compound of Formula (XVII), each R²⁹ and R⁴³is independently ethyl, isopropyl or dodecyl.

In certain embodiments of a compound of Formula (XVII), each R³⁹ ismethyl. In certain embodiments of a compound of Formula (XVII) R³ and R⁴are each hydrogen.

In certain embodiments a creatine prodrug is a compound of Formula (VI)or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof:

wherein the compound of Formula (VI) is:

wherein:

R is —CH₃ or —CD₃;

R¹⁰ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂heteroalkyl, substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl,substituted C₃₋₁₂ cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀cycloalkylalkyl, C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀heterocycloalkylalkyl, C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂heteroaryl, substituted C₅₋₁₂ heteroaryl, C₆₋₂₀ arylalkyl, substitutedC₆₋₂₀ arylalkyl, C₆₋₂₀ heteroarylalkyl, substituted C₆₋₂₀heteroarylalkyl, —C(O)R⁵, —C(O)OR⁵, —C(O)(NR³R⁴), —C(R³R⁴)—C(O)OR²²,—C(R³R⁴)—(O)C(O) R²², —C(R³R⁴)—(O)C(O)—OR²²;

R¹¹ and R¹² are each independently hydrogen or —OR¹³; or R¹¹ and R¹² areeach —C(O)R⁵, with the proviso that both R¹¹ and R¹² cannot be hydrogenR¹³ is independently hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl,C₁₋₁₂ heteroalkyl, substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl,substituted C₃₋₁₂ cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀cycloalkylalkyl, C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀heterocycloalkylalkyl, C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂heteroaryl, substituted C₅₋₁₂ heteroaryl, C₆₋₂₀ arylalkyl, substitutedC₆₋₂₀ arylalkyl, C₆₋₂₀ heteroarylalkyl, substituted C₆₋₂₀heteroarylalkyl —CH(OR⁵), —C(O)R⁵, —C(O)OR⁵ or —C(O)(NR³R⁴);

each R³ and R⁴ is independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl;

R⁵ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl;

R²³ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₅₋₁₂ cycloalkyl,substituted C₅₋₁₂ cycloalkyl, C₅₋₁₂ aryl, and C₅₋₁₂ substituted aryl,—C(O)—OR²² or —C(O)—R²²;

R²² is C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl; and

n is an integer from 1 to 2.

In certain embodiments of a compound of Formula (VI), each R⁵, R¹⁰ andR²² is independently C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₃₋₇cycloalkyl, substituted C₃₋₇ cycloalkyl, C₅₋₇ aryl or substituted C₅₋₇aryl.

In certain embodiments of a compound of Formula (VI), each R⁵, R¹⁰ andR²² is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl,neopentyl, dodecyl, 1,1-dimethoxyethyl, 1,1-diethoxyethyl, phenyl,4-methoxyphenyl, benzyl, phenethyl, styryl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, 2-pyridyl, 3-pyridyl or 4-pyridyl.

In certain embodiments of a compound of Formula (VI), each R⁵, R¹⁰ andR²² is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl,sec-pentyl, neopentyl, dodecyl, 1,1-diethoxyethyl, phenyl, cyclohexyl or3-pyridyl.

In certain embodiments of a compound of Formula (VI), each R⁵, R¹⁰ andR²² is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,dodecyl, tert-butyl, phenyl or cyclohexyl.

In certain embodiments of a compound of Formula (VI), each R⁵, R¹⁰ andR²² is independently ethyl, isopropyl or dodecyl.

In certain embodiments of a compound of Formula (VI), each R³ and R⁴ isindependently hydrogen.

In certain embodiments of a compound of Formula (VI), R¹¹ and R¹² areeach hydroxyl.

In certain embodiments of a compound of Formula (VI) one of R¹¹ or R¹²is hydrogen and the other is hydroxyl.

In certain embodiments of a compound of Formula (VI), each R²³ ishydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl, dodecyl,phenyl or cyclohexyl.

In certain embodiments of a compound of Formula (VI), each R²³ ismethyl.

In certain embodiments of a compound of Formula (VI), each substituentgroup is independently halogen, —NO₂, —OH, —NH₂, —CN, —CF₃, —OCF₃, ═O,C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy or substituted C₁₋₁₂alkoxy, —COOR^(10′) wherein R^(10′) is hydrogen, C₁₋₃ alkyl or—(NR^(11′))₂ wherein each R^(11′) is independently hydrogen or C₁₋₃alkyl.

In certain embodiments of a compound of Formula (VI), n is the integer1.

In still another embodiment, the compound of Formula (VI) is a compoundof Formula (XXII), Formula (XXIII), Formula (XXIV), Formula (XXV),Formula (XXVI), Formula (XXVII) or Formula (XXVIII) or apharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof;

wherein the compound of formula (XXII) is:

wherein the compound of Formula (XXIII) is:

wherein the compound of Formula (XXIV) is:

wherein the compound of Formula (XXV) is:

wherein the compound of Formula (XXVI) is:

wherein the compound of Formula (XXVII) is:

wherein the compound of Formula (XXVIII) is:

wherein R is —CH₃ or —CD₃;

R^(a) is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, tert-butyl;

R³² is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl, cyclohexyl;

—CH₂—C(O)OR⁴³, —CH₂—(O)C(O)R⁴³, —CH₂—(O)C(O)OR⁴³ or

R³⁹ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl;

each R³³ is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,tert-butyl, dodecyl, phenyl or cyclohexyl; R⁴³ is hydrogen, methyl,ethyl, n-propyl, isopropyl, tert-butyl, dodecyl, phenyl or cyclohexyl;and

R³ and R⁴ are each independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl.

In certain embodiments each R³² and R³³ is independently ethyl,isopropyl or dodecyl.

In certain embodiments R³⁹ is methyl.

In certain embodiments R³ and R⁴ are each hydrogen.

In certain embodiments a creatine prodrug is a compound of Formula (VII)or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomerthereof:

wherein the compound of Formula (VII) is:

wherein:

R is —CH₃ or —CD₃;

each R¹⁴ is independently hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂alkyl, C₁₋₁₂ heteroalkyl, substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂cycloalkyl, substituted C₃₋₁₂ cycloalkyl, C₄₋₂₀ cycloalkylalkyl,substituted C₄₋₂₀ cycloalkylalkyl, C₄₋₂₀ heterocycloalkylalkyl,substituted C₄₋₂₀ heterocycloalkylalkyl, C₅₋₁₂ aryl, substituted C₅₋₁₂aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂ heteroaryl, C₆₋₂₀ arylalkyl,substituted C₆₋₂₀ arylalkyl, C₆₋₂₀ heteroarylalkyl, substituted C₆₋₂₀heteroarylalkyl —CH(OR⁵), —C(O)R⁵, —C(O)OR⁵ or —C(O)(NR³R⁴);

each R³ and R⁴ is independently hydrogen, C₁₋₁₂ alkyl or substitutedC₁₋₁₂ alkyl; and

R⁵ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl.

In certain embodiments of a compound of Formula (VII), R⁵ is C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₃₋₇ cycloalkyl, substituted C₃₋₇ cycloalkyl,C₅₋₇ aryl or substituted C₅₋₇ aryl.

In certain embodiments of a compound of Formula (VII), R⁵ is hydrogen,methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, dodecyl,1,1-dimethoxyethyl, 1,1-diethoxyethyl, phenyl, 4-methoxyphenyl, benzyl,phenethyl, styryl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,2-pyridyl, 3-pyridyl or 4-pyridyl.

In certain embodiments of a compound of Formula (VII), R⁵ is hydrogen,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, dodecyl,1,1-diethoxyethyl, phenyl, cyclohexyl or 3-pyridyl.

In certain embodiments of a compound of Formula (VII), R⁵ is hydrogen,methyl, ethyl, n-propyl, isopropyl, dodecyl, tert-butyl, phenyl orcyclohexyl.

In certain embodiments of a compound of Formula (VII), R⁵ is ethyl,isopropyl or dodecyl.

In certain embodiments of a compound of Formula (VII), each R¹⁴ isindependently hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl.

In certain embodiments of a compound of Formula (VII), one R¹⁴ is methyland the other R¹⁴ is hydrogen.

In certain embodiments of a compound of Formula (VII), each R³ and R⁴ isindependently hydrogen.

In certain embodiments of a compound of Formula (VII), each substituentgroup is independently halogen, —NO₂, —OH, —NH₂, —CN, —CF₃, —OCF₃, ═O,C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy or substituted C₁₋₁₂alkoxy, —COOR^(10′) wherein R^(10′) is hydrogen, C₁₋₃ alkyl or—(NR^(11′))₂ wherein each R^(11′) is independently hydrogen or C₁₋₃alkyl.

In yet another embodiment, the compound of Formula (VII) is a compoundof Formula (XXIX) or a pharmaceutically acceptable salt, solvate,tautomer or stereoisomer thereof;

wherein the compound of Formula (XXIX) is:

wherein R is —CH₃ or —CD₃; and

each R³⁴ is independently hydrogen, methyl, ethyl, n-propyl, isopropyl,tert-butyl, dodecyl, phenyl or cyclohexyl.

In certain embodiments of a compound of Formula (XXIX), one R³⁴ ismethyl and the other R³⁴ is hydrogen.

Synthesis of Creatine Prodrugs

Those of ordinary skill in the art will appreciate that creatine prodrugcompounds of Formulae (I), (III), (VI), (VII), and any subgenera orspecies thereof, or a pharmaceutically acceptable salt, solvate,tautomer or stereoisomer thereof may be prepared via general syntheticmethods available in the art (e.g., Wuts and Greene, “Protective Groupsin Organic Synthesis,” John Wiley & Sons, 4th ed. 2006; Harrison et al.,“Compendium of Organic Synthetic Methods,” Vols. 1-11, John Wiley & Sons1971-2003; Larock “Comprehensive Organic Transformations,” John Wiley &Sons, 2nd ed. 2000; and Paquette, “Encyclopedia of Reagents for OrganicSynthesis,” John Wiley & Sons, 11th ed. 2003). Starting materials usefulfor preparing compounds and intermediates thereof, are commerciallyavailable or can be prepared by well-known synthetic methods.

Pharmaceutical Compositions

Pharmaceutical compositions of the invention can comprise a compound ofthe invention and a pharmaceutically acceptable vehicle. Apharmaceutical composition can comprise a therapeutically effectiveamount of compound of the invention and a pharmaceutically acceptablevehicle. In certain embodiments, a pharmaceutical composition caninclude more than one compound of the invention. Pharmaceuticallyacceptable vehicles include diluents, adjuvants, excipients, andcarriers.

Pharmaceutical compositions can be produced using standard procedures(see, e.g., “Remington's The Science and Practice of Pharmacy,” 21stedition, Lippincott, Williams & Wilcox, 2005). Pharmaceuticalcompositions may be manufactured by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions may be formulated in a conventional manner using one ormore physiologically acceptable carriers, diluents, excipients orauxiliaries, which facilitate processing of compounds disclosed hereininto preparations, which can be used pharmaceutically. Properformulation can depend, in part, on the route of administration

Pharmaceutical compositions of the invention can provide therapeuticplasma concentrations of a creatine creatine upon administration to apatient. The promoiety of a creatine prodrug can be cleaved in vivoeither chemically and/or enzymatically to release creatine. One or moreenzymes present in the intestinal lumen, intestinal tissue, blood,liver, brain or any other suitable tissue of a mammal can enzymaticallycleave the promoiety of the administered prodrugs. For example, thepromoiety can be cleaved after absorption by the gastrointestinal tract(e.g., in intestinal tissue, blood, liver or other suitable tissue of amammal). In certain embodiments, a creatine remains conjugated to thepromoiety during transit across the intestinal mucosal barrier toprovide protection from presystemic metabolism. In certain embodiments,a creatine prodrug is essentially not metabolized to release thecorresponding creatine within enterocytes, but is metabolized to theparent drug within the systemic circulation. Cleavage of the promoietyof a creatine prodrug after absorption by the gastrointestinal tract mayallow the prodrug to be absorbed into the systemic circulation either byactive transport, passive diffusion or by a combination of both activeand passive processes.

Creatine prodrugs can remain intact until after passage of the prodrugthrough a biological barrier, such as the blood-brain barrier. Incertain embodiments, prodrugs of the invention can be partially cleaved,e.g., one or more, but not all, of the promoieties can be cleaved beforepassage through a biological barrier or prior to being taken up by acell, tissue or organ.

Creatine prodrugs can remain intact in the systemic circulation and beabsorbed by cells of an organ, either passively or by active transportmechanisms. In certain embodiments, a creatine prodrug will belipophilic and can passively translocate through cellular membranes.Following cellular uptake, the prodrug can be cleaved chemically and/orenzymatically to release the corresponding creatine into the cellularcytoplasm, resulting in an increase in the intracellular concentrationof the creatine. In certain embodiments, a prodrug can be permeable tointracellular membranes such as the mitochondrial membrane, and therebyfacilitate delivery of a prodrug, and following cleavage of thepromoiety or promoieties, a creatine, to an intracellular organelle suchas mitochondria.

In certain embodiments, a pharmaceutical composition can include anadjuvant that facilitates absorption of a compound of the inventionthrough the gastrointestinal epithelia. Such enhancers can, for example,open the tight-junctions in the gastrointestinal tract or modify theeffect of cellular components, such as p-glycoprotein and the like.Suitable enhancers can include alkali metal salts of salicylic acid,such as sodium salicylate, caprylic or capric acid, such as sodiumcaprylate or sodium caprate, and the like. Enhancers can include, forexample, bile salts, such as sodium deoxycholate. Various p-glycoproteinmodulators are described in U.S. Pat. Nos. 5,112,817 and 5,643,909.Various absorption enhancing compounds and materials are described inU.S. Pat. No. 5,824,638, and U.S. Application No. 2006/0046962. Otheradjuvants that enhance permeability of cellular membranes includeresorcinol, surfactants, polyethylene glycol, and bile acids.

In certain embodiments, a pharmaceutical composition can include anadjuvant that reduces enzymatic degradation of a compound of theinvention. Microencapsulation using protenoid microspheres, liposomes orpolysaccharides can also be effective in reducing enzymatic degradationof administered compounds.

A pharmaceutical composition can also include one or morepharmaceutically acceptable vehicles, including excipients, adjuvants,carriers, diluents, binders, lubricants, disintegrants, colorants,stabilizers, surfactants, fillers, buffers, thickeners, emulsifiers,wetting agents, and the like. Vehicles can be selected to alter theporosity and permeability of a pharmaceutical composition, alterhydration and disintegration properties, control hydration, enhancemanufacturability, etc.

In certain embodiments, a pharmaceutical composition can be formulatedfor oral administration. Pharmaceutical compositions formulated for oraladministration can provide for uptake of a compound of the inventionthroughout the gastrointestinal tract or in a particular region orregions of the gastrointestinal tract. In certain embodiments, apharmaceutical composition can be formulated to enhance uptake acompound of the invention from the upper gastrointestinal tract, and incertain embodiments, from the small intestine. Such compositions can beprepared in a manner known in the pharmaceutical art and can furthercomprise, in addition to a compound of the invention, one or morepharmaceutically acceptable vehicles, permeability enhancers, and/or asecond therapeutic agent.

In certain embodiments, a pharmaceutical composition can furthercomprise a substance to enhance, modulate and/or control release,bioavailability, therapeutic efficacy, therapeutic potency, stability,and the like. For example, to enhance therapeutic efficacy a compound ofthe invention can be co-administered with one or more active agents toincrease the absorption or diffusion of the drug from thegastrointestinal tract or to inhibit degradation of the drug in thesystemic circulation. In certain embodiments, a compound of theinvention can be co-administered with active agents havingpharmacological effects that enhance the therapeutic efficacy of thecompound of the invention.

In certain embodiments, a pharmaceutical composition can furthercomprise substances to enhance, modulate and/or control release,bioavailability, therapeutic efficacy, therapeutic potency, stability,and the like. For example, to enhance therapeutic efficacy a compound ofthe invention can be co-administered with one or more active agents toincrease the absorption or diffusion of a compound of the invention fromthe gastrointestinal tract or to inhibit degradation of the drug in thesystemic circulation. In certain embodiments, a compound of theinvention can be co-administered with active agents havingpharmacological effects that enhance the therapeutic efficacy of acompound of the invention.

Pharmaceutical compositions can take the form of solutions, suspensions,emulsions, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, aerosols, sprays, suspensions or any other form suitable foruse. Pharmaceutical compositions for oral delivery may be in the form oftablets, lozenges, aqueous or oily suspensions, granules, powders,emulsions, capsules, syrups or elixirs, for example. Orally administeredcompositions may contain one or more optional agents, for example,sweetening agents such as fructose, aspartame or saccharin, flavoringagents such as peppermint, oil of wintergreen or cherry coloring agentsand preserving agents, to provide a pharmaceutically palatablepreparation. Moreover, when in tablet or pill form, the compositions maybe coated to delay disintegration and absorption in the gastrointestinaltract, thereby providing a sustained action over an extended period oftime. Oral compositions can include standard vehicles such as mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Such vehicles can be of pharmaceutical grade.For oral liquid preparations such as, for example, suspensions, elixirs,and solutions, suitable carriers, excipients or diluents include water,saline, alkyleneglycols (e.g., propylene glycol), polyalkylene glycols(e.g., polyethylene glycol) oils, alcohols, slightly acidic buffersbetween pH 4 and pH 6 (e.g., acetate, citrate, ascorbate at betweenabout 5 mM to about 50 mM), etc. Additionally, flavoring agents,preservatives, coloring agents, bile salts, acylcarnitines, and the likemay be added.

When a compound of the invention is acidic, it may be included in any ofthe above-described formulations as the free acid, a pharmaceuticallyacceptable salt, solvate, a solvate or a hydrate. Pharmaceuticallyacceptable salts substantially retain the activity of the free acid, maybe prepared by reaction with bases, and tend to be more soluble inaqueous and other protic solvents than the corresponding free acid form.In some embodiments, sodium salts of a compound of the invention areused in the above-described formulations.

Pharmaceutical compositions of the invention can formulated forparenteral administration including administration by injection, forexample, into a vein (intravenously), an artery (intraarterially), amuscle (intramuscularly), under the skin (subcutaneously or in a depotformulation), to the pericardium, to the coronary arteries or used as asolution for delivery to a tissue or organ, for example, use in acardiopulmonary bypass machine or to bathe transplant tissues or organs.Injectable compositions can be pharmaceutical compositions for any routeof injectable administration, including, but not limited to,intravenous, intrarterial, intracoronary, pericardial, perivascular,intramuscular, subcutaneous, intradermal, intraperitoneal, andintraarticular. In certain embodiments, an injectable pharmaceuticalcomposition can be a pharmaceutically appropriate composition foradministration directly into the heart, pericardium or coronaryarteries.

Pharmaceutical compositions of the invention suitable for parenteraladministration can comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous, water-miscible or non-aqueous vehicles. Pharmaceuticalcompositions for parenteral use may include substances that increase andmaintain drug solubility such as complexing agents and surface actingagents, compounds that make the solution isotonic or near physiologicalpH such as sodium chloride, dextrose, and glycerin, substances thatenhance the chemical stability of a solution such as antioxidants, inertgases, chelating agents, and buffers, substances that enhance thechemical and physical stability, substances that minimize selfaggregation or interfacial induced aggregation, substances that minimizeprotein interaction with interfaces, preservatives includingantimicrobial agents, suspending agents, emulsifying agents, andcombinations of any of the foregoing. Pharmaceutical compositions forparenteral administration can be formulated as solutions, suspensions,emulsions, liposomes, microspheres, nanosystems, and powder to bereconstituted as solutions. Parenteral preparations are described in“Remington, The Science and Practice of Pharmacy,” 21st edition,Lippincott, Williams & Wilkins, Chapter 41-42, pages 802-849, 2005.

In certain embodiments a pharmaceutical composition can be formulatedfor bathing transplantation tissue or organs before, during or aftertransit to an intended recipient. Such compositions can be used beforeor during preparation of a tissue or organ for transplant. In certainembodiments, a pharmaceutical composition can be a cardioplegic solutionadministered during cardiac surgery. In certain embodiments, apharmaceutical composition can be used, for example, in conjunction witha cardiopulmonary bypass machine to provide the pharmaceuticalcomposition to the heart. Such pharmaceutical compositions can be usedduring the induction, maintenance or reperfusion stages of cardiacsurgery (see e.g., Chang et al., Masui 2003, 52(4), 356-62; Ibrahim etal., Eur. J. Cardiothorac Surg 1999, 15(1), 75-83; von Oppell et al., JThorac Cardiovasc Surg. 1991, 102(3), 405-12; and Ji et al., J. ExtraCorpor Technol 2002, 34(2), 107-10). In certain embodiments, apharmaceutical composition can be delivered via a mechanical device suchas a pump or perfuser (see e.g., Hou and March, J Invasive Cardiol 2003,15(1), 13-7; Maisch et al., Am. J Cardiol 2001, 88(11), 1323-6; andMacris and Igo, Clin Cardiol 1999, 22 (1, Suppl 1), 136-9).

For prolonged delivery, a pharmaceutical composition can be provided asa depot preparation, for administration by implantation, e.g.,subcutaneous, intradermal or intramuscular injection. Thus, in certainembodiments, a pharmaceutical composition can be formulated withsuitable polymeric or hydrophobic materials, e.g., as an emulsion in apharmaceutically acceptable oil, ion exchange resins or as a sparinglysoluble derivative, e.g., as a sparingly soluble salt form of a compoundof the invention.

Pharmaceutical compositions of the invention can be formulated so as toprovide immediate, sustained or delayed release of a compound of Formula(I) and/or Formula (II) after administration to the patient by employingprocedures known in the art (see, e.g., Allen et al., “Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems,” 8th ed.,Lippincott, Williams & Wilkins, August 2004).

Dosage Forms

Pharmaceutical compositions of the invention can be formulated in a unitdosage form. Unit dosage form refers to a physically discrete unitsuitable as a unitary dose for patients undergoing treatment, with eachunit containing a predetermined quantity of a compound of the inventioncalculated to produce an intended therapeutic effect. A unit dosage formcan be for a single daily dose or one of multiple daily doses, e.g., 2to 4 times per day. When multiple daily doses are used, the unit dosagecan be the same or different for each dose. One or more dosage forms cancomprise a dose, which may be administered to a patient at a singlepoint in time or during a time interval.

Pharmaceutical compositions of the invention can be used in dosage formsthat provide immediate release and/or controlled release of a compoundof the invention. The appropriate type of dosage form can depend on thedisease, disorder or condition being treated, and on the method ofadministration. For example, for the treatment of acute ischemicconditions such as cardiac failure or stroke the use of an immediaterelease pharmaceutical composition or dosage form administeredparenterally may be appropriate. For treatment of chronicneurodegenerative disorders, controlled release pharmaceuticalcomposition or dosage form administered orally may be appropriate.

In certain embodiments, a dosage form can be adapted to be administeredto a patient no more than twice per day, and in certain embodiments,only once per day. Dosing may be provided alone or in combination withother drugs and may continue as long as required for effective treatmentof the disease, disorder or condition.

Pharmaceutical compositions comprising a compound of the invention canbe formulated for immediate release for parenteral administration oraladministration or by any other appropriate route of administration.

Controlled drug delivery systems can be designed to deliver a drug insuch a way that the drug level is maintained within the therapeuticwindows and effective and safe blood levels are maintained for a periodas long as the system continues to deliver the drug at a particularrate. Controlled drug delivery can produce substantially constant bloodlevels of a drug as compared to fluctuations observed with immediaterelease dosage forms. For some drugs, maintaining a constant bloodstreamand tissue concentration throughout the course of therapy is the mostdesirable mode of treatment. Immediate release of these drugs can causeblood levels to peak above the level required to elicit the desiredresponse, which wastes the drug and may cause or exacerbate toxic sideeffects. Controlled drug delivery can result in optimum therapy, and notonly can reduce the frequency of dosing, and may also reduce theseverity of side effects. Examples of controlled release dosage formsinclude dissolution controlled systems, diffusion controlled systems,ion exchange resins, osmotically controlled systems, erodable matrixsystems, pH independent formulations, gastric retention systems, and thelike.

In certain embodiments, an oral dosage form of the invention can be acontrolled release dosage form. Controlled delivery technologies canimprove the absorption of a drug in a particular region or regions ofthe gastrointestinal tract. The appropriate oral dosage form for aparticular pharmaceutical composition of the invention can depend, atleast in part, on the gastrointestinal absorption properties of thecompound of the invention, the stability of the compound of theinvention in the gastrointestinal tract, the pharmacokinetics of thecompound of the invention, and the intended therapeutic profile. Anappropriate controlled release oral dosage form can be selected for aparticular the compound of the invention. For example, gastric retentionoral dosage forms can be appropriate for compounds absorbed primarilyfrom the upper gastrointestinal tract, and sustained release oral dosageforms can be appropriate for compounds absorbed primarily form the lowergastrointestinal tract.

Certain compounds are absorbed primarily from the small intestine. Ingeneral, compounds traverse the length of the small intestine in about 3to 5 hours. For compounds that are not easily absorbed by the smallintestine or that do not dissolve readily, the window for active agentabsorption in the small intestine may be too short to provide a desiredtherapeutic effect. Gastric retention dosage forms, i.e., dosage formsthat are designed to be retained in the stomach for a prolonged periodof time, can increase the bioavailability of drugs that are most readilyabsorbed by the upper gastrointestinal tract. The residence time of aconventional dosage form in the stomach is 1 to 3 hours. Aftertransiting the stomach, there is approximately a 3 to 5 hour window ofbioavailability before the dosage form reaches the colon. However, ifthe dosage form is retained in the stomach, the drug can be releasedbefore it reaches the small intestine and will enter the intestine insolution in a state in which it can be more readily absorbed. Anotheruse of gastric retention dosage forms is to improve the bioavailabilityof a drug that is unstable to the basic conditions of the intestine(see, e.g., Hwang et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1998, 15, 243-284). To enhance drug absorption from the uppergastrointestinal tract, several gastric retention dosage forms have beendeveloped. Examples include, hydrogels (see, e.g., U.S. Application No.2003/0008007), buoyant matrices (see, e.g., U.S. Application No.2006/0013876), polymer sheets (see, e.g., U.S. Application No.2005/0249798), microcellular foams (see, e.g., U.S. Application No.2005/0202090), and swellable dosage forms (see, e.g., U.S. ApplicationNo. 2005/0019409; U.S. Pat. No. 6,797,283; U.S. Application No.2006/0045865; U.S. Application No. 2004/0219186; U.S. Pat. Nos.6,723,340; 6,476,006; 6,120,803; 6,548,083; 6,635,280; 5,780,057).Bioadhesive polymers can also provide a vehicle for controlled deliveryof drugs to a number of mucosal surfaces in addition to the gastricmucosa (see, e.g., U.S. Pat. Nos. 6,235,313; 6,207,197; U.S. ApplicationNo. 2006/0045865 and U.S. Application No. 2005/0064027). Ion exchangeresins have been shown to prolong gastric retention, potentially byadhesion.

In a swelling and expanding system, dosage forms that swell and changedensity in relation to the surrounding gastric content can be retainedin the stomach for longer than a conventional dosage form. A dosage formcan absorb water and swell to form a gelatinous outside surface andfloat on the surface of gastric content surface while maintainingintegrity before releasing a drug. Fatty materials can be added toimpede wetting and enhance flotation when hydration and swelling aloneare insufficient. Materials that release gases may also be incorporatedto reduce the density of a gastric retention dosage form. Swelling alsocan significantly increase the size of a dosage form and thereby impededischarge of the non-disintegrated swollen solid dosage form through thepylorus into the small intestine. Swellable dosage forms can be formedby encapsulating a core containing drug and a swelling agent or bycombining a drug, swelling agent, and one or more erodible polymers.

Gastric retention dosage forms can also be in the form of a folded thinsheet containing a drug and water-insoluble diffusible polymer thatopens in the stomach to its original size and shape, which issufficiently large to prevent or inhibit passage of the expanded dosagefrom through the pyloric sphincter.

Floating and buoyancy gastric retention dosage forms can be designed totrap gases within sealed encapsulated cores that can float on thegastric contents, and thereby be retained in the stomach for a longertime, e.g., 9 to 12 hours. Due to the buoyancy effect, these systems canprovide a protective layer preventing the reflux of gastric content intothe esophageal region and can also be used for controlled releasedevices. A floating system can, for example, contain hollow corescontaining drug coated with a protective membrane. The trapped air inthe cores floats the dosage from on the gastric content until thesoluble ingredients are released and the system collapses. In otherfloating systems, cores contain drug and chemical substances capable ofgenerating gases when activated. For example, coated cores, containingcarbonate and/or bicarbonate can generate carbon dioxide in the reactionwith hydrochloric acid in the stomach or incorporated organic acid inthe system. The gas generated by the reaction is retained to float thedosage form. The inflated dosage form later collapses and clears formthe stomach when the generated gas permeates slowly through theprotective coating.

Bioadhesive polymers can also provide a vehicle for controlled deliveryof drugs to a number of mucosal surfaces in addition to the gastricmucosa (see, e.g., U.S. Pat. Nos. 6,235,313; and 6,207,197). Abioadhesive system can be designed by incorporation of a drug and otherexcipients within a bioadhesive polymer. On ingestion, the polymerhydrates and adheres to the mucus membrane of the gastrointestinaltract. Bioadhesive polymers can be selected that adhere to a desiredregion or regions of the gastrointestinal tract. Bioadhesive polymerscan be selected to optimized delivery to targeted regions of thegastrointestinal tract including the stomach and small intestine. Themechanism of the adhesion is thought to be through the formation ofelectrostatic and hydrogen bonding at the polymer-mucus boundary. U.S.Application Nos. 2006/0045865 and 2005/0064027 disclose bioadhesivedelivery systems which are useful for drug delivery to both the upperand lower gastrointestinal tract.

Ion exchange resins have been shown to prolong gastric retention,potentially by adhesion.

Gastric retention oral dosage forms can be appropriately used fordelivery of drugs that are absorbed mainly from the uppergastrointestinal tract. For example, certain compounds of the inventionmay exhibit limited colonic absorption, and be absorbed primarily fromthe upper gastrointestinal tract. Thus, dosage forms that release thecompound of the invention in the upper gastrointestinal tract and/orretard transit of the dosage form through the upper gastrointestinaltract will tend to enhance the oral bioavailability of the compound ofthe invention. Other forms of creatine prodrugs disclosed herein can beappropriately used with gastric retention dosage forms.

Polymer matrices have also been used to achieve controlled release ofthe drug over a prolonged period of time. Such sustained or controlledrelease can be achieved by limiting the rate by which the surroundinggastric fluid can diffuse through the matrix and reach the drug,dissolve the drug and diffuse out again with the dissolved drug or byusing a matrix that slowly erodes, continuously exposing fresh drug tothe surrounding fluid. Disclosures of polymer matrices that function bythese methods are found, for example, in Skinner, U.S. Pat. Nos.6,210,710 and 6,217,903; 5,451,409; 5,945,125; PCT InternationalPublication No. WO 96/26718; U.S. Pat. Nos. 4,915,952; 5,328,942;5,783,212; 6,120,803; and 6,090,411.

Other drug delivery devices that remain in the stomach for extendedperiods of time include, for example, hydrogel reservoirs containingparticles (U.S. Pat. No. 4,871,548); swellablehydroxypropylmethylcellulose polymers (U.S. Pat. No. 4,871,548); planarbioerodible polymers (U.S. Pat. No. 4,767,627); plurality ofcompressible retention arms (U.S. Pat. No. 5,443,843); hydrophilicwater-swellable, cross-linked polymer particles (U.S. Pat. No.5,007,790); and albumin-cross-linked polyvinylpyrrolidone hydrogels(Park et al., J. Controlled Release 1992, 19, 131-134).

In certain embodiments, pharmaceutical compositions of the invention canbe practiced with a number of different dosage forms, which can beadapted to provide sustained release of the compound of the inventionupon oral administration. Sustained release oral dosage forms can beused to release drugs over a prolonged time period and are useful whenit is desired that a drug or drug form be delivered to the lowergastrointestinal tract. Sustained release oral dosage forms includediffusion-controlled systems such as reservoir devices and matrixdevices, dissolution-controlled systems, osmotic systems, anderosion-controlled systems. Sustained release oral dosage forms andmethods of preparing the same are well known in the art (see, forexample, “Remington's Pharmaceutical Sciences,” Lippincott, Williams &Wilkins, 21st edition, 2005, Chapters 46 and 47; Langer, Science 1990,249, 1527-1533; and Rosoff, “Controlled Release of Drugs,” 1989, Chapter2).

Sustained release oral dosage forms include any oral dosage form thatmaintains therapeutic concentrations of a drug in a biological fluidsuch as the plasma, blood, cerebrospinal fluid or in a tissue or organfor a prolonged time period. Sustained release oral dosage forms includediffusion-controlled systems such as reservoir devices and matrixdevices, dissolution-controlled systems, osmotic systems, anderosion-controlled systems. Sustained release oral dosage forms andmethods of preparing the same are well known in the art (see, forexample, “Remington's: The Science and Practice of Pharmacy,”Lippincott, Williams & Wilkins, 21st edition, 2005, Chapters 46 and 47;Langer, Science 1990, 249, 1527-1533; and Rosoff, “Controlled Release ofDrugs,” 1989, Chapter 2).

In diffusion-controlled systems, a water-insoluble polymer controls theflow of fluid and the subsequent egress of dissolved drug from thedosage form. Both diffusional and dissolution processes are involved inrelease of drug from the dosage form. In reservoir devices, a corecomprising a drug is coated with the polymer, and in matrix systems, thedrug is dispersed throughout the matrix. Cellulose polymers such asethylcellulose or cellulose acetate can be used in reservoir devices.Examples of materials useful in matrix systems include methacrylates,acrylates, polyethylene, acrylic acid copolymers, polyvinylchloride,high molecular weight polyvinylalcohols, cellulose derivates, and fattycompounds such as fatty acids, glycerides, and carnauba wax.

In dissolution-controlled systems, the rate of dissolution of the drugis controlled by slowly soluble polymers or by microencapsulation. Oncethe coating is dissolved, the drug becomes available for dissolution. Byvarying the thickness and/or the composition of the coating or coatings,the rate of drug release can be controlled. In somedissolution-controlled systems, a fraction of the total dose cancomprise an immediate-release component. Dissolution-controlled systemsinclude encapsulated/reservoir dissolution systems and matrixdissolution systems. Encapsulated dissolution systems can be prepared bycoating particles or granules of drug with slowly soluble polymers ofdifferent thickness or by microencapsulation. Examples of coatingmaterials useful in dissolution-controlled systems include gelatin,carnauba wax, shellac, cellulose acetate phthalate, and celluloseacetate butyrate. Matrix dissolution devices can be prepared, forexample, by compressing a drug with a slowly soluble polymer carrierinto a tablet form.

The rate of release of drug from osmotic pump systems is determined bythe inflow of fluid across a semipermeable membrane into a reservoir,which contains an osmotic agent. The drug is either mixed with the agentor is located in a reservoir. The dosage form contains one or more smallorifices from which dissolved drug is pumped at a rate determined by therate of entrance of water due to osmotic pressure. As osmotic pressurewithin the dosage form increases, the drug is released through theorifice(s). The rate of release is constant and can be controlled withintight limits yielding relatively constant plasma and/or bloodconcentrations of the drug. Osmotic pump systems can provide a constantrelease of drug independent of the environment of the gastrointestinaltract. The rate of drug release can be modified by altering the osmoticagent and the sizes of the one or more orifices.

The release of drug from erosion-controlled systems is determined by theerosion rate of a carrier matrix. Drug is dispersed throughout thepolymer and the rate of drug release depends on the erosion rate of thepolymer. The drug-containing polymer can degrade from the bulk and/orfrom the surface of the dosage form.

Sustained release oral dosage forms can be in any appropriate form fororal administration, such as, for example, in the form of tablets, pillsor granules. Granules can be filled into capsules, compressed intotablets or included in a liquid suspension. Sustained release oraldosage forms can additionally include an exterior coating to provide,for example, acid protection, ease of swallowing, flavor,identification, and the like.

In certain embodiments, sustained release oral dosage forms can comprisea therapeutically effective amount of a compound of the invention and apharmaceutically acceptable vehicle. In certain embodiments, a sustainedrelease oral dosage form can comprise less than a therapeuticallyeffective amount of a compound of the invention and a pharmaceuticallyeffective vehicle. Multiple sustained release oral dosage foams, eachdosage form comprising less than a therapeutically effective amount of acompound of the invention, can be administered at a single time or overa period of time to provide a therapeutically effective dose or regimenfor treating a disease in a patient associated with a dysfunction inenergy metabolism such as, for example, ischemia, oxidative stress, aneurodegenerative disease, including amyotrophic lateral sclerosis(ALS), Huntington's disease, Parkinson's disease or Alzheimer's disease,ischemic reperfusion injury, a cardiovascular disease, multiplesclerosis (MS), a psychotic disorder, a genetic disease affecting thecreatine kinase system or muscle fatigue.

Sustained release oral dosage forms of the invention can release acompound of the invention from the dosage form to facilitate the abilityof the compound of the invention to be absorbed from an appropriateregion of the gastrointestinal tract, for example, in the smallintestine or in the colon. In certain embodiments, a sustained releaseoral dosage from can release a compound of the invention from the dosageform over a period of at least about 4 hours, at least about 8 hours, atleast about 12 hours, at least about 16 hours, at least about 20 hours,and in certain embodiments, at least about 24 hours. In certainembodiments, a sustained release oral dosage form can release a compoundof the invention from the dosage form in a delivery pattern of fromabout 0 wt % to about 20 wt % in about 0 to about 4 hours, about 20 wt %to about 50 wt % in about 0 to about 8 hours, about 55 wt % to about 85wt % in about 0 to about 14 hours, and about 80 wt % to about 100 wt %in about 0 to about 24 hours. In certain embodiments, a sustainedrelease oral dosage form can release a compound of Formula (I) and/orFormula (II) from the dosage form in a delivery pattern of from about 0wt % to about 20 wt % in about 0 to about 4 hours, about 20 wt % toabout 50 wt % in about 0 to about 8 hours, about 55 wt % to about 85 wt% in about 0 to about 14 hours, and about 80 wt % to about 100 wt % inabout 0 to about 20 hours. In certain embodiments, a sustained releaseoral dosage form can release a compound of the invention from the dosageform in a delivery pattern of from about 0 wt % to about 20 wt % inabout 0 to about 2 hours, about 20 wt % to about 50 wt % in about 0 toabout 4 hours, about 55 wt % to about 85 wt % in about 0 to about 7hours, and about 80 wt % to about 100 wt % in about 0 to about 8 hours.

Sustained release oral dosage forms comprising a creatine prodrugcompound of the invention can provide a concentration of creatine in theplasma, blood or tissue of a patient over time, following oraladministration to the patient. The concentration profile of creatine canexhibit an AUC that is proportional to the dose of the correspondingcompound of the invention.

Regardless of the specific form of controlled release oral dosage formused, a compound of the invention can be released from an orallyadministered dosage form over a sufficient period of time to provideprolonged therapeutic concentrations of the compound of the invention inthe plasma and/or blood of a patient. Following oral administration, adosage form comprising a compound of the invention can provide atherapeutically effective concentration of creatine in the plasma and/orblood of a patient for a continuous time period of at least about 4hours, of at least about 8 hours, for at least about 12 hours, for atleast about 16 hours, and in certain embodiments, for at least about 20hours following oral administration of the dosage form to the patient.The continuous time periods during which a therapeutically effectiveconcentration of creatine is maintained can be the same or different.The continuous period of time during which a therapeutically effectiveplasma concentration of creatine is maintained can begin shortly afteroral administration or after a time interval.

In certain embodiments, an oral dosage for treating a disease, disorderor condition in a patient can comprise a compound of the inventionwherein the oral dosage form is adapted to provide, after a singleadministration of the oral dosage form to the patient, a therapeuticallyeffective concentration of creatine in the plasma of the patient for afirst continuous time period selected from at least about 4 hours, atleast about 8 hours, at least about 12 hours, and at least about 16hours, and at least about 20 hours.

Methods of Use

The creatine kinase (creatine-creatine phosphate) system serves a numberof functions in maintaining intracellular energy homeostasis (see e.g.,Walsh et al., J Physiol, 2001, 537, 971-978). Phosphocreatine acts as atemporal energy buffer at intracellular sites of high energytranslocation which operates when the rate of ATP utilization is greaterthan the rate of ATP production by mitochondrial respiration.Mitochondrial creatine kinase allows the high energy phosphate bond ofnewly synthesized ATP to be transferred to creatine, thus generatingphosphocreatine, which is much more stable than ATP. Phosphocreatine candiffuse throughout a cell and its high energy phosphate bond can be usedto regenerate ATP from ADP at heavy energy utilization sites where othercreatine kinase enzymes are strategically positioned. These sitesinclude membranes that engage in ion transport, axonal regions involvedin transporting material along microtubules to and from presynapticendings, and presynaptic endings where energy is required forneurotransmission. Neurons synthesize creatine, however the amount ofcreatine can be severely depleted during injury. As with skeletal andheart muscle, neuronal creatine stores can to some extent be increasedby oral supplementation of creatine. The creatine kinase system alsoserves as an intracellular spatial energy transport mechanism. In thisrole as an energy carrier, energy generated by the ATP-ADP system withinmitochondria is coupled to the creatine-creatine phosphate system in thecytosol, which in turn is coupled to extra-mitochondrial ATP-ADP systemsat sites of high intracellular energy transduction. Thecreatine-creatine phosphate system is also believed to act as a lowthreshold ADP sensor that maintains ATP-ADP concentration ratios insubcellular locations wherein creatine kinase is functionally coupled toATP-consuming and ATP-producing pathways. For example, it has been shownthat creatine can react with ATP derived from mitochondrial respirationin a reaction catalyzed by mitochondrial creatine kinase andfunctionally coupled to adenine nucleotide translocase, therebyresulting in an increase in local ADP concentration and the stimulationof mitochondrial respiration. The creatine kinase system is thereforeparticularly important in maintaining energy homeostasis, including ATPhomeostasis, in cells, tissues, and organs with high-energy consumptionrequirements such as neurons and muscles.

Compounds of the invention and pharmaceutical compositions of theinvention can be useful in treating of diseases, disorders or conditionsin a patient associated with a dysfunction in energy metabolism. Incertain embodiments, the dysfunction in energy metabolism comprises adepletion in intracellular ATP concentration, a decreased intracellularcreatine phosphate concentration, a decreased intracellular creatinephosphate to ATP concentration ratio, and/or a dysfunction in thecreatine kinase system in a tissue or organ affected by the disease. Incertain embodiments, a dysfunction in energy metabolism comprises adecreased intracellular ATP concentration in a tissue or organ affectedby the disease. In certain embodiments, a dysfunction in energymetabolism comprises a decreased intracellular creatine phosphateconcentration in a tissue or organ affected by the disease. In certainembodiments, the dysfunction in energy metabolism comprises adysfunction in the creatine kinase system and/or other intracellularenergy pathway in a tissue or organ affected by the disease. In certainembodiments, a disease associated with a dysfunction in energymetabolism is selected from ischemia, oxidative stress, aneurodegenerative disease, ischemic reperfusion injury, a cardiovasculardisease, multiple sclerosis, a psychotic disease, and muscle fatigue. Incertain embodiments, treating a disease comprises effecting energyhomeostasis in a tissue or organ affected by the disease.

Compounds of the invention and pharmaceutical compositions thereof canbe used to treat a disease in a patient associated with oxidative stressby administering to a patient in need of such treatment atherapeutically effective amount of a compound of the invention or apharmaceutical composition thereof. In certain embodiments, theoxidative stress is associated with ischemia or a neurodegenerativedisorder. Methods of the invention include treating an oxidativelystressed tissue or organ by contacting the tissue or organ with acompound of the invention or a pharmaceutical composition thereof.

Compounds and pharmaceutical compositions of the invention can be usefulin treating diseases, disorders or conditions in which a rapid increasein intracellular creatine levels has a therapeutic effect.

Ischemia

Compounds and pharmaceutical compositions of the invention can be usedto treat acute or chronic ischemic diseases, disorders or conditions.Ischemia is an imbalance of oxygen supply and demand in a cell, tissueor organ. Ischemia is characterized by hypoxia, including anoxia,insufficiency of metabolic substrates for normal cellular bioenergetics,and accumulation of metabolic waste. Ischemia in a tissue or organ maybe caused by a vascular insufficiency such as arteriosclerosis,thrombosis, embolism, torsion or compression, hypotension such as shockor hemorrhage, increased tissue mass (hypertrophy), increased workload(tachycardia, exercise), and/or by decreased tissue stress such ascardiac dilation. Ischemia can also result from trauma or surgicalprocedures. Depending on the severity and duration of the injury,ischemia can lead to a reversible loss of cellular function or toirreversible cell death. Different cell types have different thresholdsto ischemic injury depending, at least in part, on the cellular energyrequirements of the tissue(s) or organ(s) affected. Parenchymal cellssuch as neurons (3-4 minutes), cardiac muscles, hepatocytes, renaltubular cells, gastrointestinal epithelium (20-80 minutes) andfibroblasts, epidermis, and skeletal muscle (hours) are more susceptibleto ischemic injury than are stromal cells. A number of studies suggest acorrelation between the functional capacity of the creatine kinasesystem and ischemic tolerance of a given tissue, and indicate thatstrategies toward improving the functional capacity of the creatinekinase system may be effective for improving ischemic tolerance intissue (see e.g., Wyss and Kaddurah-Daouk, Physiological Reviews, 2000,80(3), 1107-1213, which is incorporated by reference herein in itsentirety). For example oral creatine supplementation inhibitsmitochondrial cytochrome C release and downstream caspase-3 activation,resulting in ischemic neuroprotection. Associated with inhibition ofcytochrome C release and caspase-3 activation and with neuroprotection,creatine administration inhibits ischemia-mediated ATP depletion.

Compounds and pharmaceutical compositions of the invention can be usedto treat acute or chronic ischemia. In certain embodiments, a compoundor composition can be particularly useful in acute or emergencytreatment of ischemia in tissue or organs characterized by high energydemand such as the brain, neurons, heart, lung, kidney or the intestine.

The high energy requirements compared to the low energy reserves renderthe brain particularly vulnerable to hypoxic conditions. Although thebrain constitutes only a small fraction of total body weight (about 2%),it accounts for a disproportionately large percentage of 02 consumption(about 20%). Under physiological conditions, enhanced demand for 02 israpidly and adequately compensated for by an increase in cerebral bloodflow. The longer the duration of hypoxia/ischemia, the larger and morediffuse the areas of the brain that are affected. The areas mostvulnerable to ischemic damage are the brainstem, hippocampus, andcerebral cortex. Injury progresses and eventually becomes irreversibleexcept if oxygenation is not restored. Acute cell death occurs mainlythrough necrosis but hypoxia also causes delayed apoptosis. In additionglutamate release from presynaptic neurons can further enhance Ca²⁺influx and result in catastrophic collapse in postsynaptic cells. If theischemia is not too severe, cells can suppress some functions, i.e.,protein synthesis and spontaneous electrical activity, in a processcalled penumbra, which can be reversed provided that 02 supply isresumed. However, the process of restoring oxygen levels to ischemicallystressed tissue, e.g., reperfusion, can also induce irreversible celldeath, mainly through the generation of reactive oxygen species andinflammatory cell infiltration.

The neuron is limited by its availability of energy-generatingsubstrates, being limited to using primarily glucose, ketone bodies orlactate. The neuron does not produce or store glucose or ketone bodiesand cannot survive for any significant period of time without asubstrate, which is absorbed and used directly or indirectly from thebloodstream. Thus, a constant supply of an energy-generating substratemust be present in the blood at all times in an amount sufficient tosupply the entire brain and the rest of the body with energy-generatingsubstrates. Brain cells require a concentration of about 5 mM glucose(or its equivalent) in order to maintain its optimal rate of oxidativephosphorylation to produce ATP. Nutrients enter cells by passing throughthe cell membrane. Nutrient delivery frequently relies upon mechanismsoutside the cell membranes such as oral intake, absorption, circulatorytransport and interstitial flux. Once localized in the vicinity of thecell, membrane-specific processes play a role in nutrient transportsequentially across the blood-brain barrier and then into the interiorof the cell and into various subcellular organelles. Nutrient transportis made possible by the breakdown of ATP by ATPases. Na⁺ gradientscreated by Na⁺/K⁺ ATPases can be used by cells to transport nutrientmolecules across cell membranes.

Lack of oxygen or glucose prevents or limits the ability of neurons tosynthesize ATP. The intracellular creatine/phosphocreatine system can tosome extent compensate for the lack of oxygen or glucose. Creatinekinase catalyses the synthesis of phosphocreatine from creatine innormal brain tissue. Under conditions of ATP depletion, phosphocreatinecan donate its phosphate group to ADP to resynthesize ATP. However,neuronal phosphocreatine content is limited following complete anoxia orischemia phosphocreatine is also rapidly depleted. ATP depletion isbelieved to block Na⁺/K⁺ ATPases causing neurons to depolarize and losemembrane potential.

Depleted oxygen levels have several other consequences on cellularbioenergetics and function that can ultimately lead to cell death. Forexample, dysfunctional bioenergetics also involves impaired calciumhomeostasis. The regulation of calcium plays a central role in theproper functioning and survival of neurons. Calcium pumps, located oncell membranes, use ATP to transport calcium ions out of the neuron.Proper activity of the calcium pump is essential in the maintenance ofneuronal, mitochondrial, and endoplasmic reticulum homeostasis.Alterations in calcium pump function modulate enzyme activity within acell and also play a critical role in triggering the mitochondrialpermeability transition, which may lead to cell death. For example,intracellular Ca²⁺ metabolism is believed to contribute to cell death inAlzheimer's disease. For example, under conditions of oxidative stress,the production of oxygen free radicals exceeds endogenous free radicalprotective mechanisms. This impairs neuronal metabolism and function bydirect free radical damage to important cellular biomolecules includingmembrane lipids, nucleic acids, and functional proteins; and bymodulation of critical signal transduction pathways. Neural function isdependent upon transmission of electrical impulses between cells. Thisactivity relies upon the precise actions of multiple membrane proteinseach suspended in a phospholipid bilayer. The optimal activity of thisdynamic membrane microenvironment depends upon the exact status andchemical composition of the lipid constituents. Lacking the appropriatephospholipid environment, cell channel proteins, enzymes, and receptorsare not able to achieve sustained levels of optimal function. Inaddition, oxidative stress and/or abnormal methyl metabolism can reducethe fluidity of the membranous lipid bilayer with subsequent adverseeffects upon embedded functional proteins. Dysfunctional bioenergeticsmay also adversely affect passage of high-energy electrons along therespiratory chain.

Apoptosis refers to the energy-requiring process of programmed celldeath whereupon an individual nerve cell under appropriate circumstancesinitiates a process leading to cell death. Certain of the mechanismsdiscussed above may initiate apoptotic pathways including oxidativestress, calcium overload, cellular energy deficiency, trophic factorwithdrawal, and abnormal amyloid precursor protein processing. Inischemia, neurons in the brain tissue region that are most severelyaffected by hypoxic injury die rapidly by necrosis, whereas neuronsexposed to lesser degrees of hypoxia die by apoptosis. The shift fromnecrotic cell death to apoptotic cell death is associated withincreasing levels of intra cellular ATP. It has been shown that creatinesupplementation can result in a greater ability to buffer ATP levels andreduce cell death and thereby provide protection from anoxic andischemic damage (Balestrino et al., Amino Acids, 2002, 23, 221-229; andZhu et al., J Neurosci 2004, 24(26), 5909-5912, each of which isincorporated by reference herein in its entirety).

In certain embodiments, compounds and pharmaceutical compositions of theinvention can be used to treat a cardiovascular disease, includingcerebral ischemia (stroke) and myocardial ischemia (heart infarction).Ischemic heart disease, as the underlying cause of many cases of acutemyocardial infarction, congestive heart failure, arrhythmias, and suddencardiac death, is a leading cause of morbidity and mortality in allindustrialized nations. In the United States, ischemic heart diseasecauses nearly 20% of all deaths (.about.600,000 deaths each year) withmany of these deaths occurring before the patient arrives at thehospital. An estimated 1.1 million Americans will have a new orrecurrent acute myocardial infarction each year, and many survivors willexperience lasting morbidity, with progression to heart failure anddeath. As the population grows older and co-morbidities such as obesityand diabetes become more prevalent, the public health burden caused byischemic heart disease is likely to increase.

Optimal cellular bioenergetics rely on: (1) adequate delivery of oxygenand substrates to the mitochondria; (2) the oxidative capacity ofmitochondria; (3) adequate amounts of high-energy phosphate and thecreatine phosphate/ATP ratio; (4) efficient energy transfer frommitochondria to sites of energy utilization; (5) adequate localregulation of ATP/ADP ratios near ATPases; and (6) efficient feedbacksignaling from utilization sites to maintain energetic homeostasis inthe cell. Defects in these cardiac energetic pathways have been found incardiovascular diseases such as dilated and hypertrophiccardiomyopathies of various origins, cardiac conduction defects, andischemic heart diseases (Saks et al., J Physiol 2006, 571.2, 253-273;Ventura-Clapier et al., J Physiol 2003, 555.1, 1-13; and Ingwall andWeiss, Circ Res 2004, 95, 135-145, each of which is incorporated byreference herein in its entirety). A decrease in the creatinephosphate/ATP ratio is consistently reported in failing human heart andexperimental heart failure, even at moderate workloads. Creatine,creatine transporter, creatine phosphate, and ATP are significantlyreduced and the decrease in the creatine phosphate/ATP ratio is apredictor of mortality in congenital heart failures. Also, adown-regulation of creatine transporter protein expression has beenshown in experimental animal models of heart disease, as well as infailing human myocardium, indicating that the generally lowered creatinephosphate and creatine levels measured in failing hearts are related todown-regulated creatine transporter capacity.

Cardiovascular disease includes hypertension, heart failure such ascongestive heat failure or heart failure following myocardialinfarction, arrhythmia, diastolic dysfunction such as left ventriculardiastolic dysfunction, diastolic heart failure or impaired diastolicfilling, systolic dysfunction, ischemia such as myocardial ischemia,cardiomyopathy such as hypertrophic cardiomyopathy and dilatedcardiomyopathy, sudden cardiac death, myocardial fibrosis, vascularfibrosis, impaired arterial compliance, myocardial necrotic lesions,vascular damage in the heart, vascular inflammation in the heart,myocardial infarction including both acute post-myocardial infarctionand chronic post-myocardial infarction conditions, coronary angioplasty,left ventricular hypertrophy, decreased ejection fraction, coronarythrombosis, cardiac lesions, vascular wall hypertrophy in the heart,endothelial thickening, myocarditis, and coronary artery disease such asfibrinoid necrosis or coronary arteries. Ventricular hypertrophy due tosystemic hypertension in association with coronary ischemic heartdisease is recognized as a major risk factor for sudden death, postinfarction heart failure, and cardiac rupture. Patients with severe leftventricular hypertrophy are particularly susceptible to hypoxia orischemia.

Neuroprotective effects of compounds of the invention can be determinedusing animal models of cerebral ischemia such as those described, forexample, in Cimino et al., Neurotoxicol 2005, 26(5), 9929-33; Konstas etal., Neurocrit Care 2006, 4(2), 168-78; Wasterlain et al., Neurology1993, 43(11), 2303-10; and Zhu et al., J Neuroscience 2004, 24(26),5909-5912.

Ischemic Reperfusion Injury

Reperfusion injury is damage to tissue when blood supply returns to thetissue after a period of ischemia. The absence in a tissue or organ ofoxygen and nutrients from blood creates a condition in which therestoration of circulation results in inflammation and oxidative damagefrom the oxygen, rather than restoration of normal function. The damageof ischemic reperfusion injury is due in part to the inflammatoryresponse of damaged tissue. Reperfusion contributes to the ischemiccascade in the brain, which is involved in stroke and brain trauma.Repeated bouts of ischemia and reperfusion also are believed to be afactor leading to the formation and failure to heal of chronic woundssuch as pressure sores and diabetic foot ulcers (Mustoe, Am J Surgery2004, 187(5), S65-S70, which is incorporated by reference herein in itsentirety). In certain embodiments, the methods and compositions of thedisclosure can protect the muscle and organs such as, for example, theheart, liver, kidney, brain, lung, spleen and steroidogenic organs, e.g.thyroid, adrenal glands, and gonads, from damage as a result of ischemiareperfusion injury.

Ischemia followed by reperfusion is a major cause of skeletal andcardiac muscle damage in mammals. Ischemia is caused by a reduction inoxygen supplied to tissues or organs as a result of reduced blood flowand can lead to organ dysfunction. Reduced blood supply can result fromocclusion or blood diversion due to vessel thrombosis, such asmyocardial infarction, stenosis, accidental vessel injury or surgicalprocedures. Subsequent reestablishment of an adequate supply ofoxygenated blood to the tissue or organ can result in increased damage,a process known as ischemia reperfusion injury or occlusion reperfusioninjury. Complications arising from ischemia reperfusion injury includestroke, fatal or non-fatal myocardial infarction, myocardial remodeling,aneurysms, peripheral vascular disease, tissue necrosis, kidney failure,and post-surgical loss of muscle tone.

Restoration of coronary blood flow following a transient period ofischemia (reperfusion), though necessary for myocyte survival and torestore aerobic metabolism, introduces a separate series of stressesthat can exacerbate cell injury. Reactive oxygen species generatedduring reperfusion damage proteins and membrane structures withincardiomyocytes and can activate signal transduction pathways that leadto apoptosis. Adherence of leukocytes to postischemic endothelial cellscan clog capillaries and release inflammatory mediators. Uponreperfusion, the influx of activated complement, catecholamines, andother signaling molecules contained in plasma or elaborated locallywithin the myocardial wall may also influence the course of eventswithin cells of the myocardium. As with the direct consequences ofischemia, reperfusion injury is an important feature of acute coronarysyndromes. Such injury occurs both spontaneously, as a result offibrinolysis of coronary thromboses, and as a consequence offibrinolytic drugs of acute angioplasty, treatments that are nowcommonly used to open occluded vessels.

In certain embodiments, compounds of the invention and compositionsthereof can be used to treat a condition associated with ischemicreperfusion injury or reduce ischemic reperfusion injury. Ischemicreperfusion injury can be associated with oxygen deprivation, neutrophilactivation, and/or myeloperoxidase production. Ischemic reperfusioninjury can be the result of a number of disease states or can beiatrogenically induced, for example, by blood clots, stenosis orsurgery.

In certain embodiments, compounds of the invention and compositionsthereof can be used to treat stroke, a fatal or non-fatal myocardialinfarction, peripheral vascular disease, tissue necrosis, and kidneyfailure, and post-surgical loss of muscle tone resulting from ischemicreperfusion injury. In certain embodiments, the methods and compositionsof the invention reduce or mitigate the extent of ischemic reperfusioninjury.

In certain embodiments, compounds of the invention and compositionsthereof can be used to treat, reduce or prevent ischemic reperfusioninjury associated with occlusion or blood diversion due to vesselstenosis, thrombosis, accidental vessel injury or surgical procedures.

In certain embodiments, compounds of the invention and compositionsthereof can also be used to treat any other condition associated withischemic reperfusion such as myocardial infarction, stroke, intermittentclaudication, peripheral arterial disease, acute coronary syndrome,cardiovascular disease and muscle damage as a result of occlusion of ablood vessel.

In certain embodiments, compounds of the invention and compositionsthereof can be used to treat reperfusion injury associated withmyocardial infarction, stenosis, at least one blood clot, stroke,intermittent claudication, peripheral arterial disease, acute coronarysyndrome, cardiovascular disease or muscle damage as a result ofocclusion of a blood vessel.

In certain embodiments, compounds of the invention and compositionsthereof can be used in conjunction with cardiac surgery, for example, inor with cardioplegic solutions to prevent or minimize ischemia orreperfusion injury to the myocardium. In certain embodiments, themethods and compositions can be used with a cardiopulmonary bypassmachine during cardiac surgery to prevent or reduce ischemic reperfusioninjury to the myocardium.

In certain embodiments, the methods and compositions of the inventioncan protect muscle and organs such as, for example, the heart, liver,kidney, brain, lung, spleen and steroidogenic organs, e.g. thyroid,adrenal glands, and gonads, from damage as a result of ischemiareperfusion injury.

Compounds and pharmaceutical compositions of the invention can be usedto treat ischemic reperfusion injury in a tissue or organ by contactingthe tissue or organ with an effective amount of the compound orpharmaceutical composition. The tissue or organ may be in a patient oroutside of a patient, i.e., extracorporeal. The tissue or organ can be atransplant tissue or organ, and the compound or pharmaceuticalcomposition can be contacted with the transplant tissue or organ beforeremoval, during transit, during transplantation, and/or after the tissueor organ is transplanted in the recipient.

In certain embodiments, compounds or pharmaceutical compositions of theinvention can be used to treat ischemic perfusion injury caused bysurgery, such as cardiac surgery. A compound or pharmaceuticalcomposition can be administered before, during, and/or after surgery. Incertain embodiments, a compound or pharmaceutical composition of theinvention can be used to treat ischemic reperfusion injury to muscle,including cardiac muscle, skeletal muscle or smooth muscle, and incertain embodiments, to treat ischemic reperfusion injury to an organsuch as the heart, lung, kidney, spleen, liver, neuron or brain. Acompound of the invention or pharmaceutical composition thereof can beadministered before, during, and/or after surgery.

In certain embodiments, compounds of the invention or pharmaceuticalcompositions of the invention can be used to treat ischemic perfusioninjury to a muscle, including cardiac muscle, skeletal muscle, andsmooth muscle.

The efficacy of a compound of the invention for treating ischemicreperfusion injury may be assessed using animal models and in clinicaltrials. Examples of useful methods for assessing efficacy in treatingischemic reperfusion injury are disclosed, for example, in Prass et al.,J Cereb Blood Flow Metab 2007, 27(3), 452-459; Arya et al., Life Sci2006, 79(1), 38-44; Lee et al., Eur. J. Pharmacol 2005, 523(1-3),101-108; and U.S. Application No. 2004/0038891. Useful methods forevaluating transplant perfusion/reperfusion are described, for example,in Ross et al., Am J. Physiol-Lung Cellular Mol. Physiol. 2000, 279(3),L528-536.

Transplant Perfusion

In certain embodiments, compounds of the invention or pharmaceuticalcompositions thereof can be used to increase the viability of organtransplants by perfusing the organs with a compound of the invention orpharmaceutical compositions thereof. Increased creatine phosphate levelsare expected to prevent or minimize ischemic damage to an organ.Perfusing with a creatine prodrug during organ removal, followingremoval of a donor organ, during implantation, and/or following organtransplantation, can enhance the viability of the organ, especially ametabolically active organ, such as the heart or pancreas, and therebyreduce rejection rates, and/or increase the time window for organtransplants.

In certain embodiments, compounds of the invention and compositionsthereof can be used to treat, prevent or reduce ischemia reperfusioninjury in extracorporeal tissue or organs. Extracorporeal tissue ororgans are tissue or organs not in an individual (also termed ex vivo),such as in transplantation. For tissue and organ transplantation, donortissue and organs removed are also susceptible to reperfusion injuryduring removal, while in transit, during implantation and followingtransplantation into a recipient. The methods and compositions can beused to increase the viability of a transplantable tissue or organ by,for example, supplementing solutions used to maintain or preservetransplantable tissues or organs. For example, the methods andcompositions can be used to bathe the transplantable tissue or organduring transport or can be placed in contact with the transplantabletissue or organ prior to, during or after transplantation.

Neurodegenerative Diseases

Neurodegenerative diseases featuring cell death can be categorized asacute, e.g., stroke, traumatic brain injury, spinal cord injury, andchronic, e.g., amyotrophic lateral sclerosis, Huntington's disease,Parkinson's disease, and Alzheimer's disease. Although these diseaseshave different causes and affect different neuronal populations, theyshare similar impairment in intracellular energy metabolism. Forexample, the intracellular concentration of ATP is decreased, resultingin cystolic accumulation of Ca²⁺ and stimulation of formation of readilyoxygen species. Ca²⁺ and reactive oxygen species, in turn, can triggerapoptotic cell death. For these disorders, impairment of brain creatinemetabolism is also evident as reflected in decreased total creatineconcentration, creatine phosphate concentration, creatine kinaseactivity, and/or creatine transporter content (see e.g., Wyss andKaddurah-Daouk, Physiol Rev 2000, 80, 1107-1213; Tarnopolsky and Beal,Ann Neurol 2001, 49, 561-574; and Butterfield and Kanski, Mech AgeingDev 2001, 122, 945-962, each of which is incorporated by referenceherein in its entirety).

Acute and chronic neurodegenerative diseases are illnesses associatedwith high morbidity and mortality and few options are available fortheir treatment. A characteristic of many neurodegenerative diseases,which include stroke, brain trauma, spinal cord injury, amyotrophiclateral sclerosis, Huntington's disease, Alzheimer's disease, andParkinson's disease, is neuronal-cell death. Cell death occurs bynecrosis or apoptosis. Necrotic cell death in the central nervous systemfollows acute ischemia or traumatic injury to the brain or spinal cord.It occurs in areas that are most severely affected by abrupt biochemicalcollapse, which leads to the generation of free radicals andexcitotoxins. Mitochondrial and nuclear swelling, dissolution oforganelles, and condensation of chromatin around the nucleus arefollowed by the rupture of nuclear and cytoplasmic membranes and thedegradation of DNA by random enzymatic cuts. Apoptotic cell death can bea feature of both acute and chronic neurological diseases. Apoptosisoccurs in areas that are not severely affected by an injury. Forexample, after ischemia, there is necrotic cell death in the core of thelesion, where hypoxia is most severe, and apoptosis occurs in thepenumbra, where collateral blood flow reduces the degree of hypoxia.Apoptotic cell death is also a component of the lesion that appearsafter brain or spinal cord injury. In chronic neurodegenerativediseases, apoptosis is the predominant form of cell death. In apoptosis,a biochemical cascade activates proteases that destroy moleculesrequired for cell survival and others that mediate a program of celldeath. Caspases directly and indirectly contribute to the morphologicchanges of the cell during apoptosis (Friedlander, N Engl J Med 2003,348(14), 1365-75). Oral creatine supplementation has been shown toinhibit mitochondrial cytochrome C release and downstream caspase-3activation, and ATP depletion inhibition of the caspase-mediated celldeath cascades in cerebral ischemia (Zhu et al., J Neurosci 2004,24(26), 5909-5912) indicating that manipulation of the creatine kinasesystem may be effective in controlling apoptotic cell death in chronicneurodegenerative diseases.

Creatine administration shows neuroprotective effects, particularly inanimal models of Parkinson's disease, Huntington's disease, and ALS(Wyss and Schulze, Neuroscience 2002, 112(2), 243-260, which isincorporated by reference herein in its entirety) and it is recognizedthat the level of oxidative stress may be a determinant of metabolicdetermination in a variety of neurodegenerative diseases. Currenthypotheses regarding the mechanisms of creatine-mediated neuroprotectioninclude enhanced energy storage, as well as stabilization of themitochondrial permeability transition pore by octomeric conformation ofcreatine kinase. It is therefore believed that higher levels ofintracellular creatine improve the overall bioenergetic status of acell, rendering the cell more resistant to injury.

Parkinson's Disease

Parkinson's disease is a slowly progressive degenerative disorder of thenervous system characterized by tremor when muscles are at rest (restingtremor), slowness of voluntary movements, and increased muscle tone(rigidity). In Parkinson's disease, nerve cells in the basal ganglia,e.g., substantia nigra, degenerate and thereby reduce the production ofdopamine and the number of connections between nerve cells in the basalganglia. As a result, the basal ganglia is unable to smooth musclemovement and coordinate changes in posture, leading to tremor,incoordination, and slowed, reduced movement (bradykinesia) (Blandini,et al., Mol. Neurobiol. 1996, 12, 73-94).

It is believed that oxidative stress may be a factor in the metabolicdeterioration seen in Parkinson's disease tissue (Ebadi et al., ProgNeurobiol 1996, 48, 1-19; Jenner and Olanow, Ann Neurol 1998, 44 Suppl1, S72-S84; and Sun and Chen, J Biomed Sci 1998, 5, 401-414, each ofwhich is incorporated by reference herein in its entirety) and creatinesupplementation has been shown to exhibit neuroprotective effects(Matthews et al., Exp Neurol, 1999, 157, 142-149, which is incorporatedby reference herein in its entirety).

The efficacy of administering a compound of the invention for treatingParkinson's disease may be assessed using animal and human models ofParkinson's disease and clinical studies. Animal and human models ofParkinson's disease are known (see, e.g., O'Neil et al., CNS Drug Rev.2005, 11(1), 77-96; Faulkner et al., Ann. Pharmacother. 2003, 37(2),282-6; Olson et al., Am. J. Med. 1997, 102(1), 60-6; Van Blercom et al.,Clin Neuropharmacol. 2004, 27(3), 124-8; Cho et al., Biochem. Biophys.Res. Commun. 2006, 341, 6-12; Emborg, J. Neuro. Meth. 2004, 139,121-143; Tolwani et al., Lab Anim Sci 1999, 49(4), 363-71; Hirsch etal., J Neural Transm Suppl 2003, 65, 89-100; Orth and Tabrizi, MovDisord 2003, 18(7), 729-37; Betarbet et al., Bioessays 2002, 24(4),308-18; and McGeer and McGeer, Neurobiol Aging 2007, 28(5), 639-647).

Alzheimer's Disease

Alzheimer's disease is a progressive loss of mental functioncharacterized by degeneration of brain tissue, including loss of nervecells and the development of senile plaques and neurofibrillary tangles.In Alzheimer's disease, parts of the brain degenerate, destroying nervecells and reducing the responsiveness of the maintaining neurons toneurotransmitters. Abnormalities in brain tissue consist of senile orneuritic plaques, e.g., clumps of dead nerve cells containing anabnormal, insoluble protein called amyloid, and neurofibrillary tangles,twisted strands of insoluble proteins in the nerve cell.

It is believed that oxidative stress may be a factor in the metabolicdeterioration seen in Alzheimer's disease tissue with creatine kinasebeing one of the targets of oxidative damage (Pratico et al., FASEB J1998, 12, 1777-1783; Smith et al., J Neurochem 1998, 70, 2212-2215; andYatin et al., Neurochem Res 1999, 24, 427-435, each of which isincorporated by reference herein in its entirety) and studies have showna correlation between intracellular levels of creatine phosphate and theprogress of dementia (Pettegrew et al., Neurobiol Aging 1994, 15,117-132, which is incorporated by reference herein in its entirety).

The efficacy of administering a compound of the invention for treatingAlzheimer's disease may be assessed using animal and human models ofAlzheimer's disease and clinical studies. Useful animal models forassessing the efficacy of compounds for treating Alzheimer's disease aredisclosed, for example, in Van Dam and De Dyn, Nature Revs Drug Disc2006, 5, 956-970; Simpkins et al., Ann NY Acad Sci, 2005, 1052, 233-242;Higgins and Jacobsen, Behav Pharmacol 2003, 14(5-6), 419-38; Janus andWestaway, Physiol Behav 2001, 73(5), 873-86; and Conn, ed., “Handbook ofModels in Human Aging,” 2006, Elsevier Science & Technology.

Huntington's Disease

Huntington's disease is an autosomal dominant neurodegenerative disorderin which specific cell death occurs in the neostriatum and cortex(Martin, N Engl J Med 1999, 340, 1970-80, which is incorporated byreference herein in its entirety). Onset usually occurs during thefourth or fifth decade of life, with a mean survival at age onset of 14to 20 years. Huntington's disease is fatal, and there is no effectivetreatment. Symptoms include a characteristic movement disorder(Huntington's chorea), cognitive dysfunction, and psychiatric symptoms.The disease is caused by a mutation encoding an abnormal expansion ofCAG-encoded polyglutamine repeats in the protein, huntingtin. A numberof studies suggest that there is a progressive impairment of energymetabolism, possibly resulting from mitochondrial damage caused byoxidative stress as a consequence of free radical generation.Preclinical studies in animal models of Huntington's disease havedocumented neuroprotective effects of creatine administration. Forexample, neuroprotection by creatine is associated with higher levels ofcreatine phosphate and creatine and reduced lactate levels in the brain,consistent with improved energy production (see, Ryu et al.,Pharmacology & Therapeutics 2005, 108(2), 193-207, which is incorporatedby reference herein in its entirety).

The efficacy of administering a compound of the invention for treatingHuntington's disease may be assessed using animal and human models ofHuntington's disease and clinical studies. Animal models of Huntington'sdisease are disclosed, for example, in Riess and Hoersten, U.S.Application No. 2007/0044162; Rubinsztein, Trends in Genetics, 2002,18(4), 202-209; Matthews et al., J. Neuroscience 1998, 18(1), 156-63;Tadros et al., Pharmacol Biochem Behav 2005, 82(3), 574-82, and in U.S.Pat. No. 6,706,764, and U.S. Application Nos. 2002/0161049,2004/0106680, and 2007/0044162. A placebo-controlled clinical trialevaluating the efficacy of creatine supplementation to treatHuntington's disease is disclosed in Verbessem et al., Neurology 2003,61, 925-230.

Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerativedisorder characterized by the progressive and specific loss of motorneurons in the brain, brain stem, and spinal cord (Rowland andSchneider, N Engl J Med 2001, 344, 1688-1700, which is incorporated byreference herein in its entirety). ALS begins with weakness, often inthe hands and less frequently in the feet, that generally progresses upan arm or leg. Over time, weakness increases and spasticity developscharacterized by muscle twitching and tightening, followed by musclespasms and possibly tremors. The average age of onset is 55 years, andthe average life expectancy after clinical onset is 4 years. The onlyrecognized treatment for ALS is riluzole, which can extend survival byonly about three months. Oral creatine has been shown to provideneuroprotective effects in a transgenic animal model of ALS (Klivenyi etal., Nat Med 1999, 5, 347-50, which is incorporated by reference hereinin its entirety).

The efficacy of administering a compound of the invention for treatingALS may be assessed using animal and human models of ALS and clinicalstudies. Natural disease models of ALS include mouse models (motorneuron degeneration, progressive motor neuropathy, and wobbler) and thehereditary canine spinal muscular atrophy canine model (Pioro andMitsumoto, Clin Neurosci, 19954996, 3(6), 375-85). Experimentallyproduced and genetically engineered animal models of ALS can also usefulin assessing therapeutic efficacy (see e.g., Doble and Kennelu,Amyotroph Lateral Scler Other Motor Neuron Disord. 2000, 1(5), 301-12;Grieb, Folia Neuropathol. 2004, 42(4), 239-48; Price et al., Rev Neurol(Paris), 1997, 153(8-9), 484-95; and Klivenyi et al., Nat Med 1999, 5,347-50). Specifically, the SOD1-G93A mouse model is a recognized modelfor ALS. Examples of clinical trial protocols useful in assessingtreatment of ALS are described, for example, in Mitsumoto, AmyotrophLateral Scler Other Motor Neuron Disord. 2001, 2 Suppl 1, S10-S14;Meininger, Neurodegener Dis 2005, 2, 208-14; and Ludolph and Sperfeld,Neurodegener Dis. 2005, 2(3-4), 215-9.

Multiple Sclerosis

Multiple sclerosis (MS) is a multifaceted inflammatory autoimmunedisease of the central nervous system caused by an autoimmune attackagainst the isolating axonal myelin sheets of the central nervoussystem. Demyelination leads to the breakdown of conduction and to severedisease with destruction of local axons and irreversible neuronal celldeath. The symptoms of MS are highly varied with each individual patientexhibiting a particular pattern of motor, sensible, and sensorydisturbances. MS is typified pathologically by multiple inflammatoryfoci, plaques of demyelination, gliosis, and axonal pathology within thebrain and spinal cord, all of which contribute to the clinicalmanifestations of neurological disability (see e.g., Wingerchuk, LabInvest 2001, 81, 263-281; and Virley, NeruoRx 2005, 2(4), 638-649).Although the causal events that precipitate the disease are not fullyunderstood, most evidence implicates an autoimmune etiology togetherwith environmental factors, as well as specific genetic predispositions.Functional impairment, disability, and handicap are expressed asparalysis, sensory and octintive disturbances spasticity, tremor, a lackof coordination, and visual impairment, which impact on the quality oflife of the individual. The clinical course of MS can vary fromindividual to individual, but invariably the disease can be categorizedin three forms: relapsing-remitting, secondary progressive, and primaryprogressive. Several studies implicate dysfunction of creatine phosphatemetabolism with the etiology and symptoms of the disease (Minderhoud etal., Arch Neurol 1992, 49(2), 161-5; He et al., Radiology 2005, 234(1),211-7; Tartaglia et al., Arch Neurology 2004, 61(2), 201-207; Duong etal., J Neurol 2007 Apr. 20; and Ju et al., Magnetic Res Imaging 2004,22, 427-429), although creatine ingestion alone does not appear to beeffective in improving exercise capacity in individuals with MS (Lambertet al., Arch Phys Med Rehab 2003, 84(8), 1206-1210).

Assessment of MS treatment efficacy in clinical trials can beaccomplished using tools such as the Expanded Disability Status Scale(Kurtzke, Neurology 1983, 33, 1444-1452) and the MS Functional Composite(Fischer et al., Mult Scler, 1999, 5, 244-250) as well as magneticresonance imaging lesion load, biomarkers, and self-reported quality oflife (see e.g., Kapoor, Cur Opinion Neurol 2006, 19, 255-259). Animalmodels of MS shown to be useful to identify and validate potentialtherapeutics include experimental autoimmune/allergic encephalomyelitis(EAE) rodent models that simulate the clinical and pathologicalmanifestations of MS (Werkerle and Kurschus, Drug Discovery Today:Disease Models, Nervous System Disorders, 2006, 3(4), 359-367; Gijbelset al., Neurosci Res Commun 2000, 26, 193-206; and Hofstetter et al., JImmunol 2002, 169, 117-125), and nonhuman primate EAE models ('t Hart etal., Immunol Today 2000, 21, 290-297).

Psychotic Disorders

In certain embodiments, compounds of the invention or pharmaceuticalcompositions thereof can be used to treat psychotic disorders such as,for example, schizophrenia, bipolar disorder, and anxiety.

Schizophrenia

Schizophrenia is a chronic, severe, and disabling brain disorder thataffects about one percent of people worldwide, including 3.2 millionAmericans. Schizophrenia encompasses a group of neuropsychiatricdisorders characterized by dysfunctions of the thinking process, such asdelusions, hallucinations, and extensive withdrawal of the patient'sinterests from other people. Schizophrenia includes the subtypes ofparanoid schizophrenia characterized by a preoccupation with delusionsor auditory hallucinations, hebephrenic or disorganized schizophreniacharacterized by disorganized speech, disorganized behavior, and flat orinappropriate emotions; catatonic schizophrenia dominated by physicalsymptoms such as immobility, excessive motor activity or the assumptionof bizarre postures; undifferentiated schizophrenia characterized by acombination of symptoms characteristic of the other subtypes; andresidual schizophrenia in which a person is not currently suffering frompositive symptoms but manifests negative and/or cognitive symptoms ofschizophrenia (see DSM-IV-TR classifications 295.30 (Paranoid Type),295.10 (Disorganized Type), 295.20 (Catatonic Type), 295.90(Undifferentiated Type), and 295.60 (Residual Type); Diagnostic andStatistical Manual of Mental Disorders, 4^(th) Edition, AmericanPsychiatric Association, 297-319, 2005). Schizophrenia includes theseand other closely associated psychotic disorders such asschizophreniform disorder, schizoaffective disorder, delusionaldisorder, brief psychotic disorder, shared psychotic disorder, psychoticdisorder due to a general medical condition, substance-induced psychoticdisorder, and unspecified psychotic disorders (DSM-IV-TR, 4^(th)Edition, pp. 297-344, American Psychiatric Association, 2005).

Schizophrenia symptoms can be classified as positive, negative orcognitive. Positive symptoms of schizophrenia include delusion andhallucination, which can be measured using, for example, the Positiveand Negative Syndrome Scale (PANSS) (Kay et al., Schizophrenia Bulletin1987, 13, 261-276). Negative symptoms of schizophrenia include affectblunting, anergia, alogia and social withdrawal, which can be measuredfor example, using (the Scales for the Assessment of Negative Symptoms(SANS) (Andreasen, 1983, Scales for the Assessment of Negative Symptoms(SANS), Iowa City, Iowa). Cognitive symptoms of schizophrenia includeimpairment in obtaining organizing, and using intellectual knowledgewhich can be measured using the Positive and Negative SyndromeScale-cognitive subscale (PANSS-cognitive subscale) (Lindenmayer et al.,J Nerv Ment Dis 1994, 182, 631-638) or by assessing the ability toperform cognitive tasks such as, for example, using the Wisconsin CardSorting Test (see, e.g., Green et al., Am J Psychiatry 1992, 149,162-67; and Koren et al., Schizophr Bull 2006, 32(2), 310-26).

A number of studies support a correlation of schizophrenia with adysfunction in brain high energy phosphate metabolism (Fukuzako, World JBiol Psychiatry 2001, 2(2), 70-82; and Gangadhar et al., ProgNeuro-Psychopharmacology & Biological Psychiatry 2006, 30, 910-913.Patients suffering from schizophrenia exhibit lower phosphocreatinelevels in the left and right frontal regions of the brain, which arehighly correlated with hostility-suspiciousness and anxiety-depressionassessment subscales (Deicken et al., Biol Psychiatry 1994, 36(8),503-510; Volz et al., Biol Psychiatry 1998, 44, 399-404; and Volz etal., Biol Psychiatry 2000, 47, 954-961). Creatine supplementation hasaccordingly been proposed for treating schizophrenia (see e.g., Lyoo etal., Psychiatry Res: Neuroimaging 2003, 123, 87-100).

The efficacy of creatine prodrugs and pharmaceutical compositionsthereof for treating schizophrenia may be determined by methods known tothose skilled in the art. For example, negative, positive, and/orcognitive symptom(s) of schizophrenia may be measured before and aftertreatment of the patient. Reduction in such symptom(s) indicates that apatient's condition has improved. Improvement in the symptoms ofschizophrenia may be assessed using, for example, the Scale forAssessment of Negative Symptoms (SANS), Positive and Negative SymptomsScale (PANSS) (see, e.g., Andreasen, 1983, Scales for the Assessment ofNegative Symptoms (SANS), Iowa City, Iowa; and Kay et al., SchizophreniaBulletin 1987, 13, 261-276), and using Cognitive Deficits tests such asthe Wisconsin Card Sorting Test (WCST) and other measures of cognitivefunction (see, e.g., Keshavan et al., Schizophr Res 2004, 70(2-3),187-194; Rush, Handbook of Psychiatric Measures, American PsychiatricPublishing 2000; Sajatovic and Ramirez, Rating Scales in Mental Health,2nd ed, Lexi-Comp, 2003, Keefe, et al., Schizophr Res. 2004, 68(2-3),283-97; and Keefe et al., Neuropsychopharmacology, 19 Apr. 2006.

The efficacy of creatine prodrugs and pharmaceutical compositionsthereof may be evaluated using animal models of schizophrenic disorders(see e.g., Geyer and Moghaddam, in “Neuropsychopharmacology,” Davis etal., Ed., Chapter 50, 689-701, American College ofNeuropsychopharmacology, 2002). For example, conditioned avoidanceresponse behavior (CAR) and catalepsy tests in rats are shown to beuseful in predicting antipsychotic activity and EPS effect liability,respectively (Wadenberg et al., Neuropsychopharmacology, 2001, 25,633-641).

Bipolar Disorder

Bipolar disorder is a psychiatric condition characterized by periods ofextreme mood. The moods can occur on a spectrum ranging from depression(e.g., persistent feelings of sadness, anxiety, guilt, anger, isolation,and/or hopelessness, disturbances in sleep and appetite, fatigue andloss of interest in usually enjoyed activities, problems concentrating,loneliness, self-loathing, apathy or indifference, depersonalization,loss of interest in sexual activity, shyness or social anxiety,irritability, chronic pain, lack of motivation, and morbid/suicidalideation) to mania (e.g., elation, euphoria, irritation, and/orsuspiciousness). Bipolar disorder is defined and categorized in theDiagnostic and Statistical Manual of Mental Disorders, 4^(th) Ed., TextRevision (DSM-IV-TR), American Psychiatric Assoc., 200, pages 382-401.Bipolar disorder includes bipolar I disorder, bipolar II disorder,cyclothymia, and bipolar disorder not otherwise specified.

Patients with bipolar depression are shown to have impaired brain highenergy phosphate metabolism characterized by reduced levels ofphosphocreatine and creatine kinase (Kato et al., J Affect Disord 1994,31(2), 125-33; and Segal et al., Eur Neuropsychopharmacology 2007, 17,194-198) possibly involving mitochondrial energy metabolism (Stork andRenshaw, Molecular Psychiatry 2005, 10, 900-919).

Treatment of bipolar disorder can be assessed in clinical trials usingrating scales such as the Montgomery-Asberg Depression Rating Scale, theHamilton Depression Scale, the Raskin Depression Scale, Feighnercriteria, and/or Clinical Global Impression Scale Score (Gijsman et al.,Am J Psychiatry 2004, 161, 1537-1547).

Anxiety

Anxiety is defined and categorized in the Diagnostic and StatisticalManual of Mental Disorders, 4 t^(h) Ed., Text Revision (DSM-IV-TR),American Psychiatric Assoc., 200, pages 429-484. Anxiety disordersinclude panic attack, agoraphobia, panic disorder without agoraphobia,agoraphobia without history of panic disorder, specific phobia, socialphobia, obsessive-compulsive disorder, posttraumatic stress disorder,acute stress disorder, generalized anxiety disorder, anxiety disorderdue to a general medical condition, substance-induced anxiety disorder,and anxiety disorder not otherwise specified. Recent work has documenteda correlation of decreased levels of creatine/phosphocreatine in centrumsemiovale (a representative region of the cerebral white matter) withthe severity of anxiety (Coplan et al., Neuroimaging, 2006, 147, 27-39).

Useful animal models for assessing treatment of anxiety includefear-potentiated startle (Brown et al., J Experimental Psychol, 1951,41, 317-327), elevated plus-maze (Pellow et al., J Neurosci. Methods1985, 14, 149-167; and Hogg, Pharmacol Biochem Behavior 1996, 54(1),21-20), and fear-potentiated behavior in the elevated plus-maze (Korteand De Boer, Eur J Pharmacol 2003, 463, 163-175). Genetic animal modelsof anxiety are known (Toh, Eur J Pharmacol 2003, 463, 177-184) as areother animal models sensitive to anti-anxiety agents (Martin, ActaPsychiatr Scand Suppl 1998, 393, 74-80).

In clinical trials, efficacy can be evaluated using psychologicalprocedures for inducing experimental anxiety applied to healthyvolunteers and patients with anxiety disorders (see e.g., Graeff, etal., Brazilian J Medical Biological Res 2003, 36, 421-32) or byselecting patients based on the Structured Clinical interview for DSM-IVAxis I Disorders as described by First et al., Structured ClinicalInterview for DSM-IV Axis I Disorders, Patient Edition (SCIDIP), Version2. Biometrics Research, New York State Psychiatric Institute, New York,1995. Any of a number of scales can be used to evaluate anxiety and theefficacy of treatment including, for example, the Penn State WorryQuestionnaire (Behar et al., J Behav Ther Exp Psychiatr 2003, 34,25-43), the Hamilton Anxiety and Depression Scales, the SpielbergerState-Trait Anxiety Inventory, and the Liebowitz Social Anxiety Scale(Hamilton, J Clin Psychiatry 1980, 41, 21-24; Spielberger and Vagg, JPersonality Assess 1984, 48, 95-97; and Liebowitz, J Clin Psychiatry1993, 51, 31-35 (Suppl.)).

Genetic Diseases Affecting the Creatine Kinase System

The intracellular creatine pool is maintained by uptake of creatine fromthe diet and by endogenous creatine synthesis. Many tissues, especiallythe brain, liver and pancreas, contain the Na⁺—Cl⁻ dependent creatinetransport (SLC6A8), which is responsible for active creatine transportthrough the plasma membrane. Creatine biosynthesis involves the actionof two enzymes: L-arginine:glycine amidinotransferase (AGAT) andguanidinoacetate transferase (GAMT). AGAT catalyses the transfer of theamidino group of arginine to glycine to generate ornithine andguanidinoacetate. Guanidino acetate is methylated at the amidino groupby GAMT to give creatine (see e.g., Wyss and Kaddurah-Daouk, Phys Rev2000, 80, 1107-213).

In humans, two genetic errors in creatine biosynthesis and one increatine transporter are known and involve deficiencies of AGAT, GAMT,and creatine transporter (Schulze, Cell Biochem, 2003, 244(1-2), 143-50;Sykut-Cegielska et al., Acta Biochimica Polonica 2004, 51(4), 875-882).Patients with disorders of creatine synthesis have systemic depletion ofcreatine and creatine phosphate. Patients affected with AGAT deficiencycan show mental and motor retardation, severe delay in speechdevelopment, and febrile seizures (Item et al., Am J Hum Genet. 2001,69, 1127-1133). Patients affected with GAMT deficiency can showdevelopmental delay with absence of active speech, autism withself-injury, extra pyramidal symptoms, and epilepsy (Stromberger et al.,J Inherit Metab Dis 2003, 26, 299-308). Patients with creatinetransporter deficiency exhibit intracellular depletion of creatine andcreatine phosphate. The gene encoding the creatine transporter islocated on the X-chromosome, and affected male patients show mild tosevere mental retardation with affected females having a milderpresentation (Salomons et al., J. Inherit Metab Dis 2003, 26, 309-18;Rosenberg et al., Am J Hum Genet. 2004, 75, 97-105; deGrauw et al.,Neuropediatrics 2002, 33(5), 232-238; Clark et al., Hum Genet, 2006,April).

Creatine supplementation in dosages from about 350 mg to 2 g/kg bodyweight per day have been shown effective in resolving the clinicalsymptoms of AGAT or GAMT deficiencies (see e.g., Schulze, Cell Biochem,2003, 244(1-2), 143-50). However, unlike in patients with GAMT and AGATdeficiency, in patients with creatine transporter deficiency oralcreatine supplementation does not result in an increase in braincreatine levels (see Stockler-Ipsiroglu et al., in Physician's Guide tothe Treatment and Follow up of Metabolic Diseases, eds Blau et al.,Springer Verlag, 2004).

Muscle Fatigue

During high-intensity exercise, ATP hydrolysis is initially buffered bycreatine phosphate via the creatine kinase reaction (Kongas and vanBeek, 2^(nd) Int. Conf. Systems Biol 2001, Los Angeles Calif.,Omnipress, Madison, Wis., 198-207; and Walsh et al., J Physiol 2001,537.3, 971-78, each of which is incorporated by reference herein in itsentirety). During exercise, whereas creatine phosphate is availableinstantaneously for ATP regeneration, glycolysis is induced with a delayof a few seconds, and stimulation of mitochondrial oxidativephosphorylation is delayed even further. Because the creatine phosphatestores in muscle are limited, during high-intensity exercise, creatinephosphate is depleted within about 10 seconds. It has been proposed thatmuscle performance can be enhanced by increasing the muscle stores ofcreatine phosphate and thereby delay creatine phosphate depletion.Although creatine and/or creatine phosphate supplementation may improvemuscle performance in intermittent, supramaximal exercise, there is noindication that supplementation enhances endurance performance. On theother hand, intravenous injection of creatine phosphate appears toimprove exercise tolerance during prolonged submaximal exercise (Clark,J Athletic Train, 1997, 32, 45-51, which is incorporated by referenceherein in its entirety).

Muscle Strength

Dietary creatine supplementation in normal healthy individuals hasbeneficial side effects on muscle function, and as such its use byamateur and professional athletics has increased. There is evidence tosuggest that creatine supplementation can enhance overall muscleperformance by increasing the muscle store of creatine phosphate, whichis the most important energy source for immediate regeneration of ATP inthe first few seconds of intense exercise, by accelerating restorationof the creatine phosphate pool during recovery periods, and bydepressing the degradation of adenosine nucleotides and possibly alsoaccumulation of lactate during exercise (see e.g., Wyss andKaddurah-Daouk, Physiol Rev 2000, 80(3), 1107-1213).

However, in normal healthy individuals, the continuous and prolonged useof creatine fails to maintain elevated creatine and creatine phosphatein muscle (see e.g., Juhn et al., Clin J Sport Med 1998, 8, 286-297;Terjung et al., Med Sci Sports Exerc 2000, 32, 706-717; and Vandenbergheet al., J Appl Physiol 1997, 83, 2055-2063, each of which isincorporated by reference herein in its entirety), possibly as a resultof the down regulation of the creatine transporter activity and thetransporter protein content (Snow and Murphy, Mol Cell Biochem 2001,224(1-2), 169-181, which is incorporated by reference herein in itsentirety). Thus, creatine prodrugs of the invention may be used tomaintain, restore, and/or enhance muscle strength in a mammal, and inparticular a human.

The efficacy of administering a compound of the invention formaintaining, restoring, and/or enhancing muscle strength may be assessedusing animal and human models and clinical studies. Animal models thatcan be used for evaluation of muscle strength are disclosed, forexample, in Wirth et al., J Applied Physiol 2003, 95, 402-412 andTimson, J. Appl Physiol 1990, 69(6), 1935-1945. Muscle strength can beassessed in humans using methods disclosed, for example, in Oster, U.S.Application No. 2007/0032750, U.S. Application No. 2007/0012105, and/orusing other methods known to those skilled in the art.

Organ and Cell Viability

In certain embodiments, the isolation of viable brain, muscle,pancreatic or other cell types for research or cellular transplant canbe enhanced by perfusing cells and/or contacting cells with an isolationor growth media containing a creatine phosphate analog prodrug. Incertain embodiments, the viability of a tissue organ or cell can beimproved by contacting the tissue organ or, cell with an effectiveamount of a compound of the invention or pharmaceutical compositionthereof.

Diseases Related to Glucose Level Regulation

Administration of creatine phosphate reduces plasma glucose levels, andtherefore can be useful in treating diseases related to glucose levelregulation such as hyperglycemia, insulin dependent or independentdiabetes, and related diseases secondary to diabetes (U.S. ApplicationNo 2005/0256134).

The efficacy of administering a compound of the invention for treatingdiseases related to glucose level regulation may be assessed usinganimal and human models and clinical studies. Compounds can beadministered to animals such as rats, rabbits or monkeys, and plasmaglucose concentrations determined at various times (see e.g., U.S.Application No. 2003/0232793). The efficacy of compounds for treatinginsulin dependent or independent diabetes and related diseases secondaryto diabetes can be evaluated using animal models of diabetes such asdisclosed, for example, in Shafrir, “Animal Models of Diabetes,” Ed.,2007, CRC Press; Mordes et al., “Animal Models of Diabetes,” 2001,Harwood Academic Press; Mathe, Diabete Metab 1995, 21(2), 106-111; andRees and Alcolado, Diabetic Med. 2005, 22, 359-370.

Dose

Compounds of the invention) or pharmaceutically acceptable salts orpharmaceutically acceptable solvates of any of the foregoing can beadministered to treat diseases or disorders associated with adysfunction in energy metabolism.

The amount of a compound of the invention that will be effective in thetreatment of a particular disease, disorder or condition disclosedherein will depend on the nature of the disease, disorder or condition,and can be determined by standard clinical techniques known in the art.In addition, in vitro or in vivo assays may optionally be employed tohelp identify optimal dosage ranges. The amount of a compoundadministered can depend on, among other factors, the patient beingtreated, the weight of the patient, the health of the patient, thedisease being treated, the severity of the affliction, the route ofadministration, the potency of the compound, and the judgment of theprescribing physician.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a beneficial circulatingcomposition concentration range. Initial doses can also be estimatedfrom in vivo data, e.g., animal models, using techniques that are knownin the art. Such information can be used to more accurately determineuseful doses in humans. One having ordinary skill in the art canoptimize administration to humans based on animal data.

Creatine occurs naturally in the human body and is partly synthesized bythe kidney, pancreas, and liver (approximately 1-2 grams per day), andpartly ingested with food (approximately 1-5 grams per day). Cellsactively take up creatine via the creatine transporter. Within a cell,creatine kinase phosphorylates creatine to form a pool of creatinephosphate that can act as a temporal and spatial energy buffer.

Creatine, creatine phosphate, and analogs thereof can be administered ina high dose without adverse side effects. For example, creatinemonohydrate has been administered to athletes and body builders inamounts ranging from 2-3 gm/day, and creatine phosphate has beenadministered to patients with cardiac diseases by intravenous injectionup to 8 gm/day, without adverse side effects. Animals fed a dietcontaining up to 1% cyclocreatine also do not exhibit adverse effects(see, e.g., Griffiths and Walker, J. Biol. Chem. 1976, 251(7),2049-2054; Annesley et al., J Biol Chem 1978, 253(22), 8120-25; Lillieet al., Cancer Res 1993, 53, 3172-78; and Griffiths, J Biol Chem 1976,251(7), 2049-54).

In certain embodiments, a therapeutically effective dose of a compoundof the invention can comprise from about 1 mg-equivalents to about20,000 mg-equivalents of a creatine phosphate analog per day, from about100 mg-equivalents to about 12,000 mg-equivalents of creatine phosphateanalog per day, from about 1,000 mg-equivalents to about 10,000mg-equivalents of creatine phosphate analog per day, and in certainembodiments, from about 4,000 mg-equivalents to about 8,000mg-equivalents of creatine phosphate analog per day.

A dose can be administered in a single dosage form or in multiple dosagefowls. When multiple dosage forms are used the amount of compoundcontained within each dosage form can be the same or different. Theamount of a compound of the invention contained in a dose can depend onthe route of administration and whether the disease, disorder orcondition in a patient is effectively treated by acute, chronic or acombination of acute and chronic administration.

In certain embodiments an administered dose is less than a toxic dose.Toxicity of the compositions described herein can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) or the LD₁₀₀ (the dose lethal to 100% of the population).The dose ratio between toxic and therapeutic effect is the therapeuticindex. In certain embodiments, a pharmaceutical composition can exhibita high therapeutic index. The data obtained from these cell cultureassays and animal studies can be used in formulating a dosage range thatis not toxic for use in humans. A dose of a pharmaceutical compositionof the invention can be within a range of circulating concentrations infor example the blood, plasma or central nervous system, that includethe effective dose and that exhibits little or no toxicity. A dose mayvary within this range depending upon the dosage form employed and theroute of administration utilized.

During treatment, a dose and dosing schedule can provide sufficient orsteady state levels of an effective amount of a creatine phosphateanalog to treat a disease. In certain embodiments, an escalating dosecan be administered.

Administration

A compound of the invention, a pharmaceutically acceptable salt,solvate, tautomer or stereoisomer thereof or a pharmaceuticallyacceptable solvate of any of the foregoing or a pharmaceuticalcomposition of any of the foregoing can be administered by anyappropriate route. In certain embodiments, a compound of the inventioncan be administered intermittently or continuously. Examples of suitableroutes of administration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural oral, sublingual, intranasal, intracerebral, intravaginal,transdermal, rectally, inhalation or topically. Administration can besystemic or local. Administration can be bolus injection, continuousinfusion or by absorption through epithelial or mucocutaneous linings,e.g. oral mucosa, rectal, and intestinal mucosa, etc.

In certain embodiments, it may be desirable to introduce a compound ofthe invention, a pharmaceutically acceptable salt or a pharmaceuticallyacceptable solvate of any of the foregoing or a pharmaceuticalcomposition of any of the foregoing directly into the central nervoussystem by any suitable route, including intraventricular, intrathecal,and epidural injection. Intraventricular injection can be facilitated bythe use of an intraventricular catheter, for example, attached to areservoir, such as an Ommaya reservoir.

In certain embodiments, a compound of the invention, a pharmaceuticallyacceptable salt, solvate, tautomer or stereoisomer thereof or apharmaceutically acceptable solvate of any of the foregoing or apharmaceutical composition of any of the foregoing can be administeredparenterally, such as by injection, including, for example, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticualr, subcapsular, subarachnoid, intraspinal,and intrasternal injection or infusion.

A compound of the invention, a pharmaceutically acceptable salt,solvate, tautomer or stereoisomer thereof or a pharmaceuticallyacceptable solvate of any of the foregoing or a pharmaceuticalcomposition of any of the foregoing can be administered systemicallyand/or locally to a specific organ.

In certain embodiments, a compound of the invention or pharmaceuticalcomposition thereof can be administered as a single, one time dose orchronically. By chronic it is meant that the methods and compositions ofthe invention are practiced more than once to a given individual. Forexample, chronic administration can be multiple doses of apharmaceutical composition administered to an animal, including anindividual, on a daily basis, twice daily basis or more or lessfrequently, as will be apparent to those of skill in the art. In anotherembodiment, the methods and compositions are practiced acutely. By acuteit is meant that the methods and compositions of the invention arepracticed in a time period close to or contemporaneous with the ischemicor occlusive event. For example, acute administration can be a singledose or multiple doses of a pharmaceutical composition administered atthe onset of an ischemic or occlusive event such as acute myocardialinfarction, upon the early manifestation of an ischemic or occlusiveevent such as, for example, a stroke or before, during or after asurgical procedure. A time period close to or contemporaneous with anischemic or occlusive event will vary according to the ischemic eventbut can be, for example, within about 30 minutes of experiencing thesymptoms of a myocardial infarction, stroke or intermittentclaudication. In certain embodiments, acute administration isadministration within about an hour of the ischemic event. In certainembodiments, acute administration is administration within about 2hours, about 6 hours, about 10 hours, about 12 hours, about 15 hours orabout 24 hours after an ischemic event.

In certain embodiments, a compound of the invention or pharmaceuticalcomposition thereof can be administered chronically. In certainembodiments, chronic administration can include several intravenousinjections administered periodically during a single day. In certainembodiments, chronic administration can include one intravenousinjection administered as a bolus or as a continuous infusion daily,about every other day, about every 3 to 15 days, about every 5 to 10days, and in certain embodiments, about every 10 days.

Combination Therapy

In certain embodiments, a compound of the invention, a pharmaceuticallyacceptable salt, solvate, tautomer or stereoisomer thereof orpharmaceutically acceptable solvate of any of the foregoing, can be usedin combination therapy with at least one other therapeutic agent. Acompound of the invention and other therapeutic agent(s) can actadditively or, and in certain embodiments, synergistically. In someembodiments, a compound of the invention can be administeredconcurrently with the administration of another therapeutic agent, suchas for example, a compound for treating a disease associated with adysfunction in energy metabolism; treating muscle fatigue; enhancingmuscle strength and endurance; increasing the viability of organtransplants; and improving the viability of isolated cells. In someembodiments, a compound of the invention, a pharmaceutically acceptablesalt or a pharmaceutically acceptable solvate of any of the foregoingcan be administered prior to or subsequent to administration of anothertherapeutic agent, such as for example, a compound for treating adisease associated with a dysfunction in energy metabolism such asischemia, ventricular hypertrophy, a neurodegenerative disease such asALS, Huntington's disease, Parkinson's disease or Alzheimer's disease,surgery related ischemic tissue damage, and reperfusion tissue damage;treating multiple sclerosis (MS), treating a psychotic disorder such asschizophrenia, bipolar disorder or anxiety; treating muscle fatigue;enhancing muscle strength and endurance; increasing the viability oforgan transplants; and improving the viability of isolated cells.

Pharmaceutical compositions of the invention can include, in addition toone or more compounds of the invention, one or more therapeutic agentseffective for treating the same or different disease, disorder orcondition.

Methods of the invention include administration of one or more compoundsor pharmaceutical compositions of the invention and one or more othertherapeutic agents provided that the combined administration does notinhibit the therapeutic efficacy of the one or more compounds of theinvention and/or does not produce adverse combination effects.

In certain embodiments, compositions of the invention can beadministered concurrently with the administration of another therapeuticagent, which can be part of the same pharmaceutical composition ordosage form as or in a different composition or dosage form from, thatcontaining the compounds of the invention. In certain embodiments,compounds of the invention can be administered prior or subsequent toadministration of another therapeutic agent. In certain embodiments ofcombination therapy, the combination therapy comprises alternatingbetween administering a composition of the invention and a compositioncomprising another therapeutic agent, e.g., to minimize adverse sideeffects associated with a particular drug. When a compound of theinvention is administered concurrently with another therapeutic agentthat potentially can produce adverse side effects including, but notlimited to, toxicity, the therapeutic agent can advantageously beadministered at a dose that falls below the threshold at which theadverse side effect is elicited.

In certain embodiments, compounds or pharmaceutical compositions of theinvention include or can be administered to a patient together with,another compound for treating Parkinson's disease such as amantadine,benztropine, bromocriptine, levodopa, pergolide, pramipexole,ropinirole, selegiline, trihexyphenidyl or a combination of any of theforegoing.

In certain embodiments, compounds or pharmaceutical compositions of theinvention include or can be administered to a patient together with,another compound for treating Alzheimer's disease such as donepezil,galantamine, memantine, rivastigmine, tacrine or a combination of any ofthe foregoing.

In certain embodiments, compounds or pharmaceutical compositions of theinvention include or can be administered to a patient together with,another compound for treating ALS such as riluzole.

In certain embodiments, compounds or pharmaceutical compositions of theinvention include or can be administered to a patient together with,another compound for treating ischemic stroke such as aspirin,nimodipine, clopidogrel, pravastatin, unfractionated heparin,eptifibatide, a β-blocker, an angiotensin-converting enzyme (ACE)inhibitor, enoxaparin or a combination of any of the foregoing.

In certain embodiments, compounds or pharmaceutical compositions of theinvention include or can be administered to a patient together with,another compound for treating ischemic cardiomyopathy or ischemic heartdisease such as ACE inhibitors such as ramipril, captopril, andlisinopril; n-blockers such as acebutolol, atenolol, betaxolol,bisoprolol, carteolol, nadolol, penbutolol, propranolol, timolol,metoprolol, carvedilol, and aldosterone; diuretics; digitoxin or acombination of any of the foregoing.

In certain embodiments, compounds or pharmaceutical compositions of theinvention include or can be administered to a patient together with,another compound for treating a cardiovascular disease such as,blood-thinners, cholesterol lowering agents, anti-platelet agents,vasodilators, β-blockers, angiotensin blockers, digitalis and isderivatives or combinations of any of the foregoing.

In certain embodiments, compounds or pharmaceutical compositions of theinvention include or can be administered to a patient together with,another compound for treating MS. Examples of drugs useful for treatingMS include corticosteroids such as methylprednisolone; IFN-β such asIFN-β1a and IFN-β1b; glatiramer acetate (Copaxone®); monoclonalantibodies that bind to the very late antigen-4 (VLA-4) integrin(Tysabri®) such as natalizumab; immunomodulatory agents such as FTY 720sphinogosie-1 phosphate modulator and COX-2 inhibitors such as BW755c,piroxicam, and phenidone; and neuroprotective treatments includinginhibitors of glutamate excitotoxicity and iNOS, free-radicalscavengers, and cationic channel blockers; memantine; AMPA antagonistssuch as topiramate; and glycine-site NMDA antagonists (Virley, NeruoRx2005, 2(4), 638-649, and references therein; and U.S. Application No.2004/0102525).

In certain embodiments, compounds or pharmaceutical compositions of theinvention include or can be administered to a patient together with,another compound for treating schizophrenia. Examples of antipsychoticagents useful in treating schizophrenia include, but are not limited to,acetophenazine, alseroxylon, amitriptyline, aripiprazole, astemizole,benzquinamide, carphenazine, chlormezanone, chlorpromazine,chlorprothixene, clozapine, desipramine, droperidol, aloperidol,fluphenazine, flupenthixol, glycine, oxapine, mesoridazine, molindone,olanzapine, ondansetron, perphenazine, pimozide, prochlorperazine,procyclidine, promazine, propiomazine, quetiapine, remoxipride,reserpine, risperidone, sertindole, sulpiride, terfenadine,thiethylperzaine, thioridazine, thiothixene, trifluoperazine,triflupromazine, trimeprazine, and ziprasidone. Other antipsychoticagents useful for treating symptoms of schizophrenia includeamisulpride, balaperidone, blonanserin, butaperazine, carphenazine,eplavanserin, iloperidone, lamictal, onsanetant, paliperidone,perospirone, piperacetazine, raclopride, remoxipride, sarizotan,sonepiprazole, sulpiride, ziprasidone, and zotepine; serotonin anddopamine (5HT/D2) agonists such as asenapine and bifeprunox; neurokinin3 antagonists such as talnetant and osanetant; AMPAkines such as CX-516,galantamine, memantine, modafinil, ocaperidone, and tolcapone; anda-amino acids such as D-serine, D-alanine, D-cycloserine, andN-methylglycine.

In certain embodiments, compounds or pharmaceutical compositions of theinvention include or can be administered to a patient together with,another compound for treating bipolar disorder such as aripiprazole,carbamazepine, clonazepam, clonidine, lamotrigine, quetiapine,verapamil, and ziprasidone.

In certain embodiments, compounds or pharmaceutical compositions of theinvention include or can be administered to a patient together with,another compound for treating anxiety such as alprazolam, atenolol,busipirone, chlordiazepoxide, clonidine, clorazepate, diazepam, doxepin,escitalopram, halazepam, hydroxyzine, lorazepam, prochlorperazine,nadolol, oxazepam, paroxetine, prochlorperazine, trifluoperazine, andvenlafaxine.

EXAMPLES

The following examples describe in detail assays for thecharacterization of compounds of the invention and uses of compounds ofthe invention. It will be apparent to those skilled in the art that manymodifications, both to materials and methods, may be practiced withoutdeparting from the scope of the disclosure.

General Experimental

The NMR spectra of compounds were acquired at 400 or 500 MHz (¹H) at 25°C. ¹H NMR spectra were processed with 0.3 Hz line broadening unlessotherwise specified. For LC/MS analysis, a Shimadzu LCMS 2010 (Column:sepax ODS 50×2.0 mm, 5 um), an Agilent 1200 HPLC, 1956 MSD (Column:Waters XBridge C18 4.6×50 mm, 3.5 mm) Shim-pack XR-ODS 30×3.0, 2.2 um)operating in ES (+) ionization mode, Agilent 3110™ (or an Agilent ZorbaxBonus RP™, 2.1×50 mm, 3.5 m, was used. An exemplary set up was at atemperature of 50° C. and at a flow rate of 0.8 mL/min, 2 μL injection,mobile phase: A=water with 0.1% formic acid and 1% acetonitrile, mobilephase B=methanol with 0.1% formic acid; retention time given in minutes.Method details: (I) runs on a Binary Pump G1312B™ with UV/Vis diodearray detector G1315C and Agilent 6130™ mass spectrometer in positiveand negative ion electrospray mode with UV-detection at 220 and 254 nmwith a gradient of 5-95% B in a 2.5 min linear gradient (II) hold for0.5 min at 95% B (III) decrease from 95-5% B in a 0.1 min lineargradient (IV) hold for 0.29 min at 5% B. For analytical HPLC sampleanalysis, an Agilent 1200 Series™ was used equipped with a Waters HSST3™ column, 2.1×50 mm, 1.8 m, at a temperature of 60° C. and at a flowrate of 0.5 mL/min, mobile phase: A=water with 0.1% formic acid and 0.1%acetonitrile, mobile phase B=acetonitrile with 0.1% formic acid;retention time given in minutes. Melting point temperatures wererecorded using a Thomas Hoover Unimelt™ capillary melting pointapparatus. Reaction progress was monitored by thin layer chromatographyon Merck EMD 60 F254 silica gel coated glass plates using UV lightand/or treatment with iodine to visualize. Chromatographic purificationwas carried out on either a Teledyne ISCO CombiFlash Companion™ with avariable flow rate from 5-100 mL/min. The columns used were TeledyneISCO RediSep Disposable Flash Columns (4, 12, 24, 40, 80, or 120 gpre-packed silica gel). Peaks were detected by variable wavelength UVabsorption (200-360 nm). Preparative reverse phase chromatography wasaccomplished using a Gilson 215 Liquid Handler equipped with VarianModel 218 pumps operated using Chromeleon™ software. Detection wasachieved using either a Varian Pro Star™ UV-Vis or a Sedex 55™ ELSDunit. Chromatographic separations were achieved using a PhenomenexKinetex™ 5u C18 100A, Axia, 100×30 mm column at a flow rate of 28mLmin⁻¹.

The compounds tested in the bioassays, such as Compounds A, B, C, D, E,F, G, H, J, K, L, and M, correspond to the compounds exemplifed by thesynthetic procedures described herein. For example, Compound E is thecompound of Example 26, Step 5A as described in this application.

Example 1: Methods for Determination of Enzymatic Cleavage of ProdrugsIn Vitro

For creatine prodrugs, it is generally desirable that the prodrugremains intact (i.e., uncleaved) while in the systemic circulation andbe cleaved (i.e., to release the parent drug) in the target tissue. Auseful level of stability can at least in part be determined by themechanism and pharmacokinetics of the prodrug. A useful level oflability can at least in part also be determined by the pharmacokineticsof the prodrug and parent drug (e.g., creatine) in the systemiccirculation and/or in the gastrointestinal tract, if orallyadministered. In general, prodrugs that are more stable in thegastrointestinal tract (as may be assessed by stability in simulatedgastric fluid, simulated intestinal fluid, intestinal S9, pancreatin orcolonic wash assays) and are more labile in mouse plasma, rat plasma,human plasma, mouse, rat and/or human liver S9, liver microsomes, and/orhepatocyte preparations can be useful as an orally administered prodrug.In general, prodrugs that are more stable in mouse plasma, rat plasma,human plasma, mouse, rat and/or human liver S9, liver microsomes, and/orhepatocyte preparations and which are more labile in target tissue cellhomogenates or target tissue cell isolate preparations, such as brain,muscle, and Caco-2 S9 preparations, can be useful as systemicallyadministered prodrugs and/or can be more effective in delivering aprodrug to a target tissue. In general, prodrugs that are more stable indifferent pH physiological buffers can be more useful as prodrugs. Ingeneral, prodrugs that are more labile in target tissue cell homogenatesand/or target tissue cell isolate preparations, such brain, muscle andCaco-2 S9 preparations, can be intra-cellularly cleaved to release theparent drug to a target tissue. The results of tests, such as thosedescribed in this example, for determining the enzymatic or chemicalcleavage of prodrugs in vitro can be used to select prodrugs for in vivotesting.

The stabilities of prodrugs can be evaluated in one or more in vitrosystems using a variety of preparations following methods known in theart. Tissues and preparations are obtained from commercial sources(e.g., Pel-Freez Biologicals, Rogers, Ark., or GenTest Corporation,Woburn, Mass.). Experimental conditions useful for the in vitro studiesare described in Table 1. Prodrug is added to each preparation intriplicate.

TABLE 1 Standard^(a) Conditions for Prodrug In Vitro Stability andMetabolism Studies Substrate Enzyme/Protein Concentration AssayConcentration (μM) Cofactors SGF +/−0.1 mg/mL 1-10 NA pepsin SIF +/−1%w/v 1-10 NA pancreatin Plasma NA 1-10 +/− DIFP, 10 mM^(d) Blood NA 1-10+/− DIFP, 10 mM^(d) Liver 0.5 mg/mL 1-10 +/− NADPH^(b) microsomes Liveror 1 mg/mL 1-10 +/− NADPH^(b) Intestinal S9 Hepatocytes NA 1-10 NATissue NA 1-10 NA homogenate^(c) ^(a)Typical test range provided, rangemay be exceeded dependent upon intended clinical use of prodrug;^(b)NADPH generating system, e.g., 1.3 mM NADP+, 3.3 mMglucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, 3.3 mMmagnesium chloride and 0.95 mg/mL potassium phosphate, pH 7.4;^(c)Examples: brain tissue homogenate, muscle tissue homogenate;^(d)Assay can be performed with and without addition of diisopropylfluorophosphonate (DIFP, serine protease inhibitor) to determine ifdegradation is mediated by a serine protease, e.g., carboxylesterase(CES)

For preparations that contain alkaline phosphatases, prodrug is testedin the presence and absence of a phosphatase inhibitor cocktail (Sigma).Samples are incubated at 37° C. for times ranging from 30 minutes to 24hours. At each time point, samples are quenched with 50% ethanol.Baseline concentrations of prodrug are determined by adding the compounddirectly to the 50% ethanol/preparation mixture (t=0). Samples arecentrifuged at 14,000 rpm for 15 minutes, and concentrations of intactprodrug and released parent drug are determined using LC/MS/MS. Thisstability of prodrugs towards specific enzymes (e.g., peptidases, etc.)is also assessed in vitro by incubation with the purified enzyme.

Pancreatin stability studies are conducted by incubating prodrug (5 μM)with 1% (w/v) pancreatin (Sigma, P-1625, from porcine pancreas) in 0.025M Tris buffer containing 0.5 M NaCl (pH 7.5) at 37° C. The reaction isstopped by addition of 3 volumes of 50% ethanol. After centrifugation at14,000 rpm for 15 min, the supernatant is removed and analyzed byLC/MS/MS for prodrug, creatine, and creatinine.

For determination of stability in simulated gastric fluid (SGF), prodrug(10 μM) is incubated in SGF (0.2% NaCl w/v, 0.7% HCl v/v, pH1.2) withand without addition of pepsin (3.2 g of purified pepsin per liter withan activity of 800-2500 units per mg of protein) at 37° C. At selectedtime points (e.g., 0, 15, 30, 60 and 120 min) 50 μL aliquots are removedand neutralized by addition of 50 μL of 0.1M sodium bicarbonatesolution, followed by 150 μL of ice-cold acetonitrile. Samples arecentrifuged at 4,000×g for 15 min at 4° C., and the supernatants areremoved and analyzed for prodrug, creatine and creatinine concentrationsby LC-MSMS (Table 2).

TABLE 2 Stability of Prodrugs Simulated Gastric Fluid (SGF) with andwithout Pepsin Compound of T_(1/2) (min) Compound ID Formula (+) pepsin(−) pepsin A III 117 >360 B III 133 >360 C III >360 >360 D III >360 >360E III >360 >360 F III >360 >360 G III >360 >360

For determination of stability in simulated intestinal fluid (SIF),prodrug (10 μM) is incubated in SIF (0.68% KH₂PO4 w/v, 0.86% NaOH v/v,pH 6.8) with and without addition of pancreatin (1% w/v) at 37° C. Atselected time points (e.g., 0, 15, 30, 60 and 120 min) 50 L aliquots areremoved and the reactions are terminated by addition of 150 μL ofice-cold acetonitrile. Samples are centrifuged at 4,000×g for 15 min at4° C., and the supernatants are removed and analyzed for prodrug,creatine and creatinine concentrations by LC-MSMS (Table 3).

TABLE 3 Stability of Prodrugs Simulated Intestinal Fluid (SIF) with andwithout Pancreatin Compound of T_(1/2) (min) Compound ID Formula (+)pepsin (−) pepsin A III >360 >360 B III >360 >360 C III >360 >360 DIII >360 >360 E III >360 >360 F III >360 >360 G III >360 >360

To determine stability in Caco-2 homogenate S9, Caco-2 cells are grownfor 21 days prior to harvesting. Culture medium is removed and cellmonolayers are rinsed and scraped off into ice-cold 10 mM sodiumphosphate/0.15 M potassium chloride, pH 7.4. Cells are lysed bysonication at 4° C. using a probe sonicator. Lysed cells are thentransferred into 1.5 mL centrifuge vials and centrifuged at 9,000 g for20 min at 4° C. The resulting supernatant (Caco-2 cell homogenate S9fraction) is aliquoted into 0.5 mL vials and stored at −80° C. untilused.

For stability studies, prodrug (5 μM) is incubated in Caco-2 homogenateS9 fraction (0.5 mg/mL in 0.1M Tris buffer, pH 7.4) at 37° C. Triplicatesamples are quenched at each time point with 50% ethanol. The initial(t=0) concentration of prodrug is determined by adding 5 μM prodrugdirectly to a 50% ethanol/Caco-2 homogenate mixture. Samples aresubjected to LC/MS/MS analysis to determine concentrations of prodrug,creatine and creatinine.

To determine prodrug stability in mouse, rat, human or plasma from otherspecies, prodrugs (10 μM) or positive controls (10 μM, propantheline orprocaine) are incubated in undiluted plasma. Duplicate samples ofprodrugs and controls are prepared and analyzed. Stock solutions ofprodrugs are prepared in DMSO (10 mM) and diluted to 0.1 mM in pH 7.4phosphate buffer to prepare spiking solutions. The prodrug spikingsolutions are aliquoted (10 μL) into 96-well plates. Pre-warmed (37° C.)plasma (90 μL) is added to the wells designated for 5, 15, 30, 45 and 60min time points; for t=0 min the quench solution (400 μL acetonitrile)is added directly to the prodrug containing well followed by 90 μL ofpre-warmed plasma. At 5, 15, 30, 45 and 60 min 400 μL aliquots ofacetonitrile are added to the wells to stop the reaction. Afterquenching, the plates are shaken for 10 min (600 rpm) and thencentrifuged at 5500 g for 15 min. Aliquots (50 μL) are transferred tothe analysis plate and diluted with 100 μL ultrapure water (Millipore)for LC-MSMS quantitation of prodrug concentrations, and in some casescreatine and/or creatinine. Chromatography columns (e.g., Atlantis HILICSilica, Gemini C-18, Ultimate XB-C18) are selected based upon thelipophilicity and polarity of each prodrug. LC-MSMS platforms (e.g.,Sciex API4000, Sciex API6500) are also selected based on therequirements of each prodrug. The stabilities of the compounds of thepresent disclosure (prodrugs) in mouse plasma incubations is shown inTable 4 and stability in human plasma incubations is shown in Table 5.

TABLE 4 Stability of Selected Compounds in Mouse Plasma IncubationsCompound Compound of % Remaining ID Formula 5 min 60 min T_(1/2) (min) AIII 102 94.0 >180 B III 110 96.0 >180 C III 85.0 114 >180 D III 124118 >180 E III 100 89.0 >180 F III 103 88.2 118 G III 101 91.1 >180 HIII 96.6 94.5 >180 J VI 53.8 <LOD 3.32 K VI 37.2 0.08 NC L I M I 97.9101 >180 LOD = Limit of detection Concentration of compounds (prodrugs)and positive control: 10 μM Duplicative samples per time point obtained

TABLE 5 Stability of Selected Compounds in Human Plasma IncubationsCompound Compound of % Remaining ID Formula 5 min 60 min T_(1/2) (min) AIII 105 88.8 >180 B III 97.4 87.8 >180 C III 86.5 119 131 D III 100 109138 E III 101 75.6 154 F III 92.7 68.1 >180 G III 115 90.8 >180 H III114 109 >180 J VI 56.4 0.01 4.32 K VI 42.5 0.05 3.16 M I 96.1 91.6 >180Concentration of compounds (prodrugs) and positive control: 10 μMDuplicative samples per time point obtained

For liver microsomal stability studies, prodrug or positive control(testosterone, propafenone, diclofenac, 7-ethoxycoumarin or propranolol)are incubated (in duplicate) at 5 μM in hepatic or intestinal fractionsfrom mouse, human, dog, monkey and/or rat. Incubations are conducted at37° C. in the presence or absence of NADPH regenerating system toindicate whether metabolism proceeds via an NADPH requiring enzyme (i.e.P450s, FMOs, NADPH-P450 reductase or other oxidase enzymes). Duplicatesamples of prodrugs and controls are prepared and analyzed. Stocksolutions of prodrugs are prepared in DMSO (10 mM) and diluted to 0.05mM in a mixture of 25% MeOH/pH 7.4 phosphate buffer to prepare spikingsolutions. The prodrug spiking solutions are aliquoted (10 μL) into96-well plates. Pre-warmed (37° C.) microsome solution (80 μL) is addedto the wells designated for 5, 15, 30, 45 and 60 min time points andincubated for 10 min before initiating the reaction with 10 μL of NADPHregenerating solution. For t=0 min the quench solution (300 μLacetonitrile) is added directly to the prodrug containing well followedby the microsome solution and NADPH soluton. Incubations of the prodrugsin heat-inactivated fractions or buffer are conducted to differentiateenzymatic from non-enzymatic degradation. At specified time points(e.g., 0, 5, 10, 20, 30 and 60 min), samples are taken and terminatedwith an equal volume of cold acetonitrile containing a suitable internalstandard (e.g., labetalol, tolbutamide). After quenching, the plates arecentrifuged at 4000 g for 20 min. Aliquots (100 μL) are transferred tothe analysis plate and diluted with 400 μL ultrapure water (Millipore)for LC-MSMS quantitation of prodrug concentrations, and in some casescreatine and/or creatinine. Chromatography columns (e.g., ACE 5 Phenyl,Phenomenex C18 Synergi Hydro-RP, Atlantis HILIC Silica) are selectedbased upon the lipophilicity and polarity of each prodrug. LC-MSMSplatforms (e.g., Sciex API4000, Sciex API6500) are also selected basedon the requirements of each prodrug. The metabolic stability of thecompounds of the present disclosure of in mouse liver microsomalincubations is shown in Table 6 and human liver microsomal incubationsis shown in Table 7.

TABLE 6 Metabolic Stability of Selected Compounds in Mouse LiverMicrosomal Incubations Compound % Remaining % Remaining Compound of (w/NADPH) (No NADPH) in vivo Clint ID Formula 5 min 30 min 30 min T_(1/2)(min) (ml/min/kg) A III 109 112 111 >90 <71 B III 109 103 99.0 >90 <71 CIII 109 112 103 >90 <71 D III 89.3 69.5 91 34.7 183 E III 89.1 26.0 15.620.0 407 F III 111 94.7 309 >90 <71 G III 124 96.9 223 >90 <71 H III 126107 125 >145 <38 J VI 25.3 0.10 1.3 4.7 1160 L I 76.6 53.5 72.3 33.2 165M I NA 41.5 52.6 19.6 280 Concentration of compounds (prodrugs) andpositive control: 10 μM Duplicative samples per time point obtained

TABLE 7 Metabolic Stability of Selected Compounds in Human LiverMicrosomal Incubations Compound % Remaining % Remaining Compound of (w/NADPH) (No NADPH) in vivo Clint ID Formula 5 min 30 min 30 min T_(1/2)(min) (ml/min/kg) A III 114.2 99.2 89.0 >90 <21 B III 96.8 85.5 91 >90<21 C III 112.8 76.2 103 74.4 25 D III 83.6 91.9 108 >90 51 E III 103.882.1 106 >90 <21 F III 102.8 90.8 85.0 >90 <21 G III 111.2 83.0 103 >90<21 H III 66.5 64.9 72.4 130 9.60 J VI 38.9 1.80 5.0 11.2 111 L I 64.548.1 61.1 20.9 59.8 M I 64.1 38.8 56.6 21.5 57.9 Concentration ofcompounds (prodrugs) and positive control: 10 μM Duplicative samples pertime point obtained

For S9 stability studies, prodrug (5 μM) is incubated in mouse, human,dog, monkey and/or rat liver or intestinal S9 homogenate (0.5 mg/mL in0.1M potassium phosphate buffer, pH 7.4, 1 mM NADPH) at 37° C.Incubations are conducted in the presence or absence of NADPHregenerating system to indicate whether metabolism proceeds via an NADPHrequiring enzyme (i.e. P450s, FMOs, NADPH-P450 reductase or otheroxidase enzymes). Triplicate samples are quenched at each time pointwith 50% ethanol. The initial (t=0) concentration of prodrug isdetermined by adding 5 μM prodrug directly to a 50% ethanol/S9homogenate mixture. Samples are subjected to LC/MS/MS analysis todetermine concentrations of prodrug, creatine, and creatinine.

For hepatocyte stability studies, prodrug (5 μM) is incubated withplated hepatocytes (e.g., mouse, rat, human). Fresh hepatocytes arereceived (Life Technologies) plated in a 12-well format with overlay(except rat which has no overlay). Upon receipt, shipping media isremoved immediately and replaced with 1 mL pre-warmed culture medium.Cells are acclimated overnight at 37° C. with 5% CO2 atmosphere. Mediais aspirated from the plate and replaced with 1 mL fresh mediacontaining prodrug (5 μM) or solvent control (0.0125% DMSO). Samples(triplicate) are incubated at 37° C. in 5% CO2 atmosphere for 0, 0.25,0.5, 0.75, 1, 2 and 4 hr. Extra wells with solvent control are includedfor production of calibration curves and measurement of background. Atthe selected time point, media is removed and frozen. Cells are washedtwice with cold PBS. 0.5 mL cold 70% acetonitrile containing internalstandard is added to each well and cells are gently removed from theplate by scraping. The recovered cells suspended in the organic solutionare aspirated into a vial and frozen at −80° C. For analysis, cellsolutions in 70% ACN are removed from the freezer, defrosted andvortexed. 500 μL water is added to each tube and the samples arevortexed again. Tubes are centrifuged at 13000 rpm for 10 minutes at 4°C. Cell supernatants and the original recovered media are removed andanalyzed by LC-MS/MS for determination of prodrug, creatine, andcreatinine.

Three buffers are used to determine the chemical stability of prodrug:(1) 0.1M potassium phosphate, 0.5 M NaCl, pH 2.0, (2) 0.1 M Tris-HCl,0.5 M NaCl, pH 7.4, and (3) 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0. Prodrug(5 μM) is added to each buffer in triplicate. Samples are quenched ateach time point with 50% ethanol. The initial (t=0) concentration ofprodrug is determined by adding 5 μM prodrug directly to a 50%ethanol/pH Buffer mixture. Samples are subjected to LC/MS/MS analysis todetermine concentrations of prodrug, creatine and creatinine.

Example 2: In Vitro Determination of Release of Creatine from Prodrugs

For assessment of the ability of prodrugs to release creatine, and torelease creatine preferentially to unwanted cyclization to creatinine,d3-labeled (deuterium labeled methyl group) prodrugs are incubated withliver homogenates (e.g., mouse, human) specially prepared to preserveN-reductase activity. The use of d3-labelled prodrugs is essential inorder to distinguish prodrug-derived creatine (d3-creatine) from highconcentrations of endogenous (non-labeled) creatine. Incubations (37°C.) are performed in 100 mM potassium phosphate buffer, pH 6.0 tooptimize N-reductase activity. Prodrugs are tested at finalconcentrations of 20 M and 200 M. Approximately 4-5 mg of homogenate isused for each reaction. Co-factor (NADH) is included at a finalconcentration of 1 mM. Benzamidoxime (500 μM final concentration) isused as a positive control for N-reductase activity. N-reductaseactivity is confirmed by conversion of benzamidoxime into benzamidine.Negative controls include incubations without NADH (to assessNADH-independent prodrug cleavage) and incubations without liverhomogenate (but with NAPD) to assess non-enzymatic prodrug cleavageunder the conditions of the assay. Prodrug incubations are prepared byaddition of 10 μL of prodrug stock solution (400 or 4000 μM, 40% DMSO inwater) to 100 μL of the potassium phosphate buffer, followed by additionof 70 μL of liver homogenate. Reaction is initiated by addition of 20 μLof NADH solution (10 mM), or 20 μL water for (−)NADH negative controls.At selected time points (e.g., 0, 30, 60 and 180 min) 50 μL aliquots areremoved and the reactions are terminated by addition of 150 μL ofice-cold acetonitrile (80% ACN/20% water) stop solution. The samples arecentrifuged at 15890×g for 10 minutes at 4° C., followed by transfer ofthe supernatants for storage at 40 C pending LC-MSMS analysis. Samplesupernatants are analyzed by LC-MSMS (HILIC column) for determination ofd3-prodrug, d3-creatine and d3-creatinine levels (Table 8).

TABLE 8 In Vitro Release of d3-Creatine and d3-Creatinine fromd3-Prodrugs in Mouse Liver Homogenate (MLH) Incubations CompoundCompound of Incubation Concentration (μM) at 120 min ID FormulaConditions Prodrug d3-Creatine d3-Creatinine A III Prodrug +MLH 195 45.4ND +NADH Negative Control 254 15.0 ND (No NADH) Negative Control 3610.29 ND (No MLH) E III Prodrug +MLH 4.70 39.1 ND +NADH Negative ControlND 10.0 ND (No NADH) Negative Control 240 ND ND (No MLH) F III Prodrug+MLH 157 9.68 19.9 +NADH Negative Control 176 2.25 4.52 (No NADH)Negative Control 259 ND ND (No MLH) ND = not determined

Example 3: In Vitro Determination of Caco-2 Cellular Permeability ofProdrugs

The passive permeability of creatine prodrugs is assessed in vitro usingstandard methods well known in the art (see, e.g., Stewart, et al.,Pharm. Res., 1995, 12, 693). For example, passive permeability can beevaluated by examining the flux of a prodrug across a cultured polarizedcell monolayer (e.g., Caco-2 cells).

Caco-2 cells obtained from continuous culture (passage less than 28) areseeded at high density onto Transwell polycarbonate filters. Cells aremaintained with DMEM/10% fetal calf serum+0.1 mM nonessential aminoacids+2 mM L-Gln, 5% CO₂/95% O₂, 37° C. until the day of the experiment.Permeability studies are conducted at pH 6.5 apically (in 50 mM MESbuffer containing 1 mM CaCl₂), 1 mM MgCl2, 150 mM NaCl, 3 mM KCl, 1 mMNaH₂PO₄, 5 mM glucose) and pH 7.4 basolaterally (in Hank's balanced saltsolution containing 10 mM HEPES) in the presence of efflux pumpinhibitors (250 μM MK-571, 250 μM verapamil, 1 mM Ofloxacin). Insertsare placed in 12 or 24 well plates containing buffer and incubated for30 min at 37° C. Prodrug (100 μM, 250 μM, 300 μM or 500 μM) is added tothe apical or basolateral compartment (donor) and concentrations ofprodrug and/or released parent drug (creatine) in the oppositecompartment (receiver) are determined at intervals over 1 hour usingLC/MS/MS. Values of apparent permeability (P_(app)) are calculated usingthe equation:

P _(app) =V _(r)(dC/dt)/(AC _(o))

where V_(r) is the volume of the receiver compartment in mL; dC/dt isthe total flux of prodrug and parent drug (μM/s), determined from theslope of the plot of concentration in the receiver compartment versustime; C_(o) is the initial concentration of prodrug in μM; and A is thesurface area of the membrane in cm². In certain embodiments, prodrugswith significant transcellular permeability exhibit a value of P_(app)of ≥1×10⁻⁶ cm/s, in certain embodiments, a value of P_(app) of ≥1×10⁻⁵cm/s, and in certain embodiments a value of P_(app) of ≥5×10⁻⁵ cm/s.

Example 4: Uptake by Caco-2 and HEK-2 Cells

Caco-2 or HEK Peaks are seeded onto poly-lysine coated 24-well plasticcell culture plates at 250,000 and 500,000 cells/well, respectively.Cells are incubated overnight at 37° C. Prodrug is added to each well in1 mL fresh media. Each concentration of prodrug is tested in triplicate.Media only is added to the control wells. At each time point, cells arewashed four times in Hank's Balanced Salt Solution. Cells are lysed andcompound is extracted by adding 200 μL 50% ethanol to each well for 20minutes at room temperature. Aliquots of the ethanol solution are movedto a 96-well V-bottom plate and centrifuged at 5,700 rpm for 20 minutesat 4° C. Supernatant is analyzed by LC/MS/MS to determine theconcentration of prodrug, creatine, and/or creatinine.

Example 5: Expression of SMVT in Mammalian Cells

Sodium dependent multivitamin transporter (SMVT; product of the SLC5A6gene) was subcloned into a plasmid that allows for inducible expressionby tetracycline (TREX plasmid, Invitrogen Inc., Carlsbad Calif.). TheSMVT expression plasmid was transfected into a human embryonic kidney(HEK) cell line and stable clones were isolated by G418 selection andflow activated cell sorting (FACS). Biotin uptake in a SMVT-HEK cellclone was used for validation. SMVT-HEK/TREX cells were plated in96-well plates at 100,000 cells/well at 37° C. for 24 hours andtetracycline (1 μg/mL) was added to each well for an additional 24 hoursto induce SMVT transporter expression. Radiolabeled ³H-biotin(.about.100,000 cpm/well) was added to each well. Plates were incubatedat room temperature for 10 min. Excess ³H-biotin was removed and cellswere washed three times with a 96-well plate washer with cold assaybuffer. Scintillation fluid was added to each well, and the plates weresealed and counted in a 96-well plate-based scintillation counter.

Similar methods can be used to prepare HEK cells expressing othertransporters or other cell lines expressing SMVT or other transporters.

The GenBank accession number for human SMVT is NM.021095, which isincorporated by reference herein. Reference to the SMVT transporterincludes the amino acid sequence described in or encoded by the GenBankreference number NM.021095, and, allelic, cognate and induced variantsand fragments thereof retaining essentially the same transporteractivity. Usually such variants show at least 90% sequence identity tothe exemplary GenBank nucleic acid or amino acid sequence. Substratesfor SMVT are compounds containing a free carboxylic acid and a shortalkyl chain, e.g., C₁₋₆ alkyl, ending in a cyclic or branched group.Example of SMVT substrates include biotin, pantothenic acid, and4-phenylbutyric acid.

Example 6: Competition Assays Using SMVT

To determine if a creatine prodrug binds the SMVT transporter, acompetition binding assay was developed. This assay measures howdifferent concentrations of a test compound block the uptake of aradiolabeled substrate such as biotin or pantothenic acid. Thehalf-maximal inhibitory concentration (IC₅₀) for inhibition of transportof a substrate by a test compound is an indication of the affinity ofthe test compound for the SMVT transporter. If the test compound bindsSMVT competitively with the radiolabeled substrate, less of theradiolabeled substrate is transported into the HEK cells. For testcompounds that do not interact with SMVT in a manner competitive withsubstrates the curve remains an essentially flat line, i.e., there is nodose response seen. The amount of radiolabeled substrate taken up by thecells is measured by lysing the cells and measuring the radioactivecounts per minute. Competition binding studies are performed as follows.SMVT-HEK/TREX cells are plated in 96-well plates at 100,000 cells/wellat 37° C. for 24 hours and tetracycline (1 Gg/mL) is added to each wellfor an additional 24 hours to induce SMVT transporter expression.Radiolabeled ³H-biotin (.about.100,000 cpm/well) is added to each wellin the presence and absence of various concentrations of unlabeledbiotin or pantothenic acid in duplicate or triplicate. Plates areincubated at room temperature for 10 min. Excess ³H-biotin is removedand cells are washed three times using a 96-well plate washer with coldassay buffer. Scintillation fluid is added to each well, and the platesare sealed and counted in a 96-well plate-based scintillation counter.Data is graphed and analyzed using non-linear regression analysis withPrism Software (GraphPad, Inc., San Diego, Calif.).

Example 7: Treatment of HEK SMVT Cells with Creatine Prodrugs

Uptake of unlabeled creatine prodrugs is measured in HEK cells stablyexpressing SMVT. Cells are plated at a density of 250,000 cells/well inpolylysine coated 24-well tissue culture plates. Twenty-four hours latercells are treated with tetracycline (1 μg/ml) to induce SMVT expressionor left untreated. The following day (approximately 48 hours afterseeding), the assay is performed. Creatine prodrugs (0.1 mM finalconcentration) are added to a buffered saline solution (HBSS), and 0.5mL of each test solution is added to each well. Cells are allowed totake up the test compounds for 1 or 3 hours. Test solution is aspiratedand cells washed 4 times with ice-cold HBSS. Cells are then lysed with a50% ethanol solution (0.2 mL/well) at room temperature for 15 minutes.The lysate is centrifuged at 5477×g for 15 minutes at 4° C. to removecell debris. The concentration of creatine prodrugs and creatine in thecell is determined by analytical LC/MS/MS. Transporter specific uptakeis determined by comparison with control cells lacking transporterexpression.

Example 8: Effect of Treatment on the Creatine Kinase System

HEK cells expressing SMVT are treated with buffer, a creatine prodrug(100 μM), creatine (100 μM) or creatine analog (100 μM), for a specifiedtime period according to the protocol of Example 6. Following treatment,the intracellular concentrations of the creatine prodrug, creatinephosphate, ATP, and creatine and/or creatine analog are measured byanalytical LC/MS/MS.

Example 9: Restoration of Cellular Energy Homeostasis Following SodiumAzide Treatment

An adaptation of the methods described by Weinstock and Shoham, NeuralTransm. 2004, 111(3), 347-66, is used to evaluate the protective effectson intracellular energy homeostasis of compounds of the invention.

The HEK TREX SMVT cell line is seeded at 250 k per well in a 24-wellpolylysine coated tissue culture plate. The next day, cells are treatedwith doxycycline (1 μg/mL) to express the SMVT transporter, which isrequired for efficient uptake of the creatine prodrug, e.g., a compoundof the invention, tested. The cells are incubated and assayed on thefollowing day. Cells are washed twice with HBSS buffer lacking glucose.Cells are then incubated for 20 mM at 37° C. in a 5% CO₂ incubator inthe same buffer with or without sodium azide. A typical range of sodiumazide used in these experiments is from 1 mM to 9 mM. After this time,300 μM of a prodrug of a creatine analog is added to the cells or thecells are left untreated. In some experiments, creatine is used as acomparison. The cells are incubated for an additional 20 min and thenwashed with buffer. Samples are extracted for 15 min with 50% ethanoland processed for LC/MS/MS to detect the creatine prodrug, creatine, andATP levels. Increased creatine phosphate and ATP levels in sodium azidetreated cells following exposure to a creatine prodrug indicates thatthe prodrug is capable of restoring cellular energy homeostasis.

Example 10: Protection Against 3-Nitropropionic Acid Induced Toxicity

An adaptation of the methods described by Brouillet et al., J. Neurochem2005, 95(6), 1521-40, is used to evaluate the protective effects onintracellular energy homeostasis of compounds of the invention.

The rat cardiomyoblast cell line H9c2 is obtained from ATCC (#CRL-1446).A 20 mM stock solution of 3-nitropropionic acid (3-NP) is preparedimmediately before use in normal media (DMEM/High glucose (4.5 g/L)/10%FBS/6 mM L-glutamine/PSF) and the pH is adjusted to 7.4 by dropwiseaddition of 1N sodium hydroxide. A 40 mM stock solution of a creatineprodrug, e.g. a compound of the invention, is prepared in DMSO, andcreatine is dissolved directly in serum-free media at 10 mM.

To measure the extent of cellular protection provided by the creatineprodrug and/or a creatine analog against 3-NP toxicity, H9c2 cells areplated in 96-well clear-bottom black tissue culture plates at 10K cellsper well in normal media and incubated overnight at 37° C. The followingday the media is removed and replaced with serum-free media containingserial dilutions of a creatine prodrug or creatine. The plates areincubated at 37° C. for 2 hours. Media is then removed by aspiration andreplaced with normal media containing various concentrations of 3-NP andthe plates incubated at 37° C. for an additional 20 hours. To determinethe number of viable cells in each well, an equal volume ofCellTiter-Glo reagent (Promega) is added and mixed for 10 minutes on aplate shaker at room temperature. Luminescence is measured by readingthe plates in a luminometer. The luminescence produced in this assay isproportional to the amount of ATP present, and directly relates to thenumber of metabolically active cells.

Increased viability of cells exposed to 3-NP and a creatine prodrugcompared to that of cells exposed to 3-NP and creatine indicates thatthe creatine prodrug has the capacity to maintain cellular energyhomeostasis.

Example 11: Pharmacokinetics of Creatine Prodrugs Following ColonicAdministration in Rats

Sustained release oral dosage forms, which release drug slowly overperiods of about 6 to about 24 hours, generally release a significantproportion of the dose within the colon. Thus, drugs suitable for use insuch dosage forms should be colonically absorbed. This experiment isperformed to assess the uptake and resultant levels of a creatineprodrug and creatine in a biological fluid such as the plasma/blood orcerebrospinal fluid (CSF), following intracolonic administration of acorresponding creatine prodrug, such as a compound of the invention andthereby determine the suitability of a creatine prodrug for use in anoral sustained release dosage faun. Bioavailability of a creatineprodrug and creatine following co-administration of the creatine prodrugcan be calculated relative to oral administration and/or to colonicadministration of the creatine prodrug.

Step A: Administration Protocol

Rats are obtained commercially and are pre-cannulated in both theascending colon and the jugular vein. Animals are conscious at the timeof the experiment. All animals are fasted overnight and until 4 hourspost-dosing of a creatine prodrug. The creatine prodrug is administeredas a solution (in water or other appropriate solvent and vehicles)directly into the colon via the cannula at a dose equivalent to about 1mg to about 200 mg of the creatine prodrug per kg body weight. Bloodsamples (0.3 mL) are obtained from the jugular cannula at intervals over8 hours and are immediately quenched with sodium metabisulfite or otherappropriate antioxidant to prevent oxidation of the creatine prodrug.Blood samples can be further quenched with methanol/perchloric acid toprevent post-sampling hydrolysis of the creatine prodrug. Blood samplesare analyzed as described below. Samples can also be taken from the CSFor other appropriate biological fluid.

Step B: Sample Preparation for Colonically Absorbed Prodrug

Methanol/perchloric acid (300 μL) is added to blank 1.5 mL Eppendorftubes. Rat blood (300 μL) is collected into EDTA tubes containing 75 μLof sodium metabisulfite at different times and vortexed to mix. A fixedvolume of blood (100 μL) is immediately added into the Eppendorf tubeand vortexed to mix. Ten microliters of a standard stock solution of thecreatine prodrug (0.04, 0.2, 1, 5, 25, and 100 μg/mL) and 10 μL of the10% sodium metabisulfite solution are added to 80 μL of blank rat bloodto make up a final calibration standard (0.004, 0.02, 0.1, 0.5, 2.5, and10 μg/mL). Methanol/perchloric acid (300 μL of 50/50) is then added intoeach tube followed by the addition of 20 μL of p-chlorophenylalanine.The samples are vortexed and centrifuged at 14,000 rpm for 10 min. Thesupernatant is analyzed by LC/MS/MS.

Step C: LC/MS/MS Analysis

An API 4000 LC/MS/MS spectrometer equipped with Agilent 1100 binarypumps, a CTC HTS-PAL autosampler, and a Zorbax XDB C8 4.6×150 mm columnis used during the analysis. Appropriate mobile phases can be used suchas, for example, (A) 0.1% formic acid, and (B) acetonitrile with 0.1%formic acid. Appropriate gradient conditions can be used such as, forexample: 5% B for 0.5 min, then to 98% B in 3 min, maintained at 98% Bfor 2.5 min, and then returned to 2% B for 2 min. A TurbolonSpray sourceis used on the API 4000. The analysis is done in an appropriate ion modeand the MRM transition for each analyte is optimized using standardsolution. 5 μL of each sample is injected. Non-compartmental analysis isperformed using WinNonlin software (v.3.1 Professional Version,Pharsight Corporation, Mountain View, Calif.) on individual animalprofiles. Summary statistics on major parameter estimates is performedfor C_(max) (peak observed concentration following dosing), T_(max)(time to maximum concentration is the time at which the peakconcentration is observed), AUC_((0-t)) (area under the serumconcentration-time curve from time zero to last collection time,estimated using the log-linear trapezoidal method), AUC_((0-.infn.))(area under the blood concentration time curve from time zero toinfinity, estimated using the log-linear trapezoidal method to the lastcollection time with extrapolation to infinity), and t_(1/2),z (terminalhalf-life).

The pharmacokinetic parameters of the creatine prodrug and creatinefollowing colonic administration of the corresponding creatine prodrugare determined and compared to those obtained following an equivalentcolonic dose of the creatine prodrug. Maximum concentrations of thecreatine prodrug and creatine in the blood (C_(max) values) and the areaunder blood concentration versus time curve (AUC) values afterintra-colonic dosing of a creatine prodrug that are higher than thoseachieved for colonic administration of the corresponding creatineprodrug indicate that the prodrug provides enhanced colonicbioavailability.

Example 12: Pharmacokinetics of a Creatine Prodrug Following Intravenousor Oral Administration to Rats

A creatine prodrug is administered as an intravenous bolus injection orby oral gavage to groups of four to six adult male Sprague-Dawley rats(about 250 g). Animals are conscious at the time of the experiment. Whenorally administered, the creatine prodrug is administered as an aqueoussolution (or as a solution of another appropriate solvent optionallyincluding appropriate vehicles) at an appropriate creatine prodrug doseequivalent per kg body weight. Blood samples (0.3 mL) are obtained via ajugular vein cannula at intervals over 8 hours following oral dosing.Blood is quenched immediately using, for example, acetonitrile with 1%formic acid and then is frozen at ±80° C. until analyzed. Samples mayalso be taken form the CSF or other appropriate biological fluid.

Three hundred (300) μL of 0.1% formic acid in acetonitrile is added toblank 1.5 mL tubes. Rat blood (300 μL) is collected at different timesinto tubes containing EDTA and vortexed to mix. A fixed volume of blood(100 μL) is immediately added into the tube and vortexed to mix. Tenmicroliters of a creatine prodrug standard stock solution (0.04, 0.2, 1,5, 25, and 100 μg/mL) is added to 90 μL of blank rat blood quenched with300 μL of 0.1% formic acid in acetonitrile. Then, 20 μL ofp-chlorophenylalanine is added to each tube to make a final calibrationstandard (0.004, 0.02, 0.1, 0.5, 2.5, and 10 μg/mL). Samples arevortexed and centrifuged at 14,000 rpm for 10 min. The supernatant isanalyzed by LC/MS/MS.

An API 4000 LC/MS/MS spectrometer equipped with Agilent 1100 binarypumps, a CTC HTS-PAL autosampler, and a Phenomenex Synergihydro-RP4.6×30 mm column are used in the analysis. Appropriate mobile phases andgradient conditions are used for the analysis. The analysis is done inthe appropriate ion mode and the MRM transition for each analyte isoptimized using standard solutions. Five (5) μL of each sample isinjected. Non-compartmental analysis is performed using WinNonlin (v.3.1Professional Version, Pharsight Corporation, Mountain View, Calif.) onindividual animal profiles. Summary statistics on major parameterestimates is performed for C_(max) (peak observed concentrationfollowing dosing), T_(max) (time to maximum concentration is the time atwhich the peak concentration was observed), AUC_((0-t)) (area under theserum concentration-time curve from time zero to last collection time,estimated using the log-linear trapezoidal method), AUC_((0-.infin)),(area under the serum concentration time curve from time zero toinfinity, estimated using the log-linear trapezoidal method to the lastcollection time with extrapolation to infinity), and t_(1/2) (terminalhalf-life).

The oral bioavailability (F (%) of the creatine prodrug is determined bycomparing the area under the creatine prodrug concentration vs timecurve (AUC) following oral administration with the AUC of the creatineprodrug concentration vs time curve following intravenous administrationof the creatine prodrug on a dose normalized basis.

Samples can also be obtained from the CSF and the pharmacokinetics ofthe creatine prodrug and creatine determined. Higher levels of creatineprodrug and/or creatine can indicate that the prodrug has the ability tobe translocated across the blood-brain barrier.

Similar studies on the pharmacokinetics of a creatine prodrug can beperformed in other animals including but not limited to dogs, monkeys,and human.

Example 13: Use of Animal Models to Assess the Efficacy of CreatineProdrugs for Treating Amyotrophic Lateral Sclerosis

A murine model of SOD1 mutation-associated ALS has been developed inwhich mice express the human superoxide dismutase (SOD) mutationglycine.fwdarw.alanine at residue 93 (SOD1). These SOD1 mice exhibit adominant gain of the adverse property of SOD, and develop motor neurondegeneration and dysfunction similar to that of human ALS (Gurney etal., Science 1994, 264(5166), 1772-1775; Gurney et al., Ann. Neurol.1996, 39, 147-157; Gurney, J. Neurol. Sci. 1997, 152, S67-73; Ripps etal., Proc Natl Acad Sci U.S.A. 1995, 92(3), 689-693; and Bruijn et al.,Proc Natl Acad Sci U.S.A. 1997, 94(14), 7606-7611). The SOD1 transgenicmice show signs of posterior limb weakness at about 3 months of age anddie at 4 months. Features common to human ALS include astrocytosis,microgliosis, oxidative stress, increased levels ofcyclooxygenase/prostaglandin, and as the disease progresses, profoundmotor neuron loss.

Studies are performed on transgenic mice overexpressing human Cu/Zn-SODG93A mutations (B6SJL-TgN(SOD1-G93A) 1 Gur) and non-transgenic B6/SJLmice and their wild litter mates. Mice are housed on a 12-hr day/lightcycle and (beginning at 45 d of age) allowed ad libitum access to eithertest compound-supplemented chow or as a control, regular formula coldpress chow processed into identical pellets. Genotyping can be conductedat 21 days of age as described in Gurney et al., Science 1994,264(5166), 1772-1775. The SOD1 mice are separated into groups andtreated with a test compound or serve as controls.

The mice are observed daily and weighed weekly. To assess health statusmice are weighed weekly and examined for changes inlacrimation/salivation, palpebral closure, ear twitch and pupillaryresponses, whisker orienting, postural and righting reflexes and overallbody condition score. A general pathological examination is conducted atthe time of sacrifice.

Motor coordination performance of the animals can be assessed by one ormore methods known to those skilled in the art. For example, motorcoordination can be assessed using a neurological scoring method. Inneurological scoring, the neurological score of each limb is monitoredand recorded according to a defined 4-point scale: 0=normal reflex onthe hind limbs (animal splays its hind limbs when lifted by its tail);1=abnormal reflex of hind limbs (lack of splaying of hind limbs whenanimal is lifted by the tail); 2=abnormal reflex of limbs and evidenceof paralysis; 3=lack of reflex and complete paralysis; and 4=inabilityto right when placed on the side in 30 seconds or found dead. Theprimary end point is survival with secondary end points of neurologicalscore and body weight. Neurological score observations and body weightare made and recorded five days per week. Data analysis is performedusing appropriate statistical methods.

The rotarod test evaluates the ability of an animal to stay on arotating dowel allowing evaluation of motor coordination andproprioceptive sensitivity. The apparatus is a 3 cm diameter automatedrod turning at, for example, 12 rounds per min. The rotarod testmeasures how long the mouse can maintain itself on the axle withoutfalling. The test can be stopped after an arbitrary limit of, forexample, 120 sec. If the animal falls before 120 sec, the performance isrecorded and two additional trials are performed. The mean time of 3trials is calculated. A motor deficit is indicated by a decrease ofwalking time.

In the grid test, mice are placed on a grid (length: 37 cm, width: 10.5cm, mesh size: 1×1 cm²) situated above a plane support. The number oftimes the mice put their paws through the grid is counted and serves asa measure for motor coordination.

The hanging test evaluates the ability of the animal to hang on a wire.The apparatus is a wire stretched horizontally 40 cm above a table. Theanimal is attached to the wire by its forepaws. The time needed by theanimal to catch the string with its hind paws is recorded (60 sec max)during three consecutive trials.

Electrophysiological measurements (EMG) can also be used to assess motoractivity condition. Electromyographic recordings are performed using anelectromyography apparatus. During EMG monitoring the mice areanesthetized. The measured parameters are the amplitude and the latencyof the compound muscle action potential (CMAP). CMAP is measured ingastrocnemius muscle after stimulation of the sciatic nerve. A referenceelectrode is inserted near the Achilles tendon and an active needleplaced at the base of the tail. A ground needle is inserted on the lowerback of the mice. The sciatic nerve is stimulated with a single 0.2 msecpulse at supramaximal intensity (12.9 mA). The amplitude (mV) and thelatency of the response (ms) are measured. The amplitude is indicativeof the number of active motor units, while distal latency reflects motornerve conduction velocity.

The efficacy of test compounds can also be evaluated using biomarkeranalysis. To assess the regulation of protein biomarkers in SOD1 miceduring the onset of motor impairment, samples of lumbar spinal cord(protein extracts) are applied to ProteinChip Arrays with varyingsurface chemical/biochemical properties and analyzed, for example, bysurface enhanced laser desorption ionization time of flight massspectrometry. Then, using integrated protein mass profile analysismethods, data is used to compare protein expression profiles of thevarious treatment groups. Analysis can be performed using appropriatestatistical methods.

Example 14: Clinical Trials to Assess the Efficacy of Creatine Prodrugsfor Treating Parkinson's Disease

The following clinical study may be used to assess the efficacy of acreatine prodrug in treating Parkinson's disease. Patients withidiopathic PD fulfilling the Queen Square Brain Bank criteria (Gibb etal., J Neurol Neurosurg Psychiatry 1988, 51, 745-752) with motorfluctuations and a defined short duration GABA analog response (1.5-4hours) are eligible for inclusion. Clinically relevant peak dosedyskinesias following each morning dose of their current medication area further pre-requisite. Patients are also required to have been stableon a fixed dose of treatment for a period of at least one month prior tostarting the study. Patients are excluded if their current drug regimeincludes slow-release formulations of L-Dopa, COMT inhibitors,selegiline, anticholinergic drugs or other drugs that could potentiallyinterfere with gastric absorption (e.g. antacids). Other exclusioncriteria include patients with psychotic symptoms or those onantipsychotic treatment patients with clinically relevant cognitiveimpairment, defined as MMS (Mini Mental State) score of less than 24(Folstein et al., J Psychiatr Res 1975, 12, 189-198), risk of pregnancy,Hoehn & Yahr stage 5 in off-status, severe, unstable diabetes mellitus,and medical conditions such as unstable cardiovascular disease ormoderate to severe renal or hepatic impairment. Full blood count, liver,and renal function blood tests are taken at baseline and aftercompletion of the study.

A randomized, double-blind, and cross-over study design is used. Thepharmacokinetics of a creatine prodrug and released creatine can beassessed by determining the blood concentrations at appropriate timeintervals. Creatine levels in the brain can also be determinednon-invasively by magnetic resonance spectroscopy (MRS).

For clinical assessment, motor function is assessed using UPDRS (UnitedParkinson's Disease Rating Scale) motor score and BrainTest (Giovanni etal., J Neurol Neurosurg Psychiatry 1999, 67, 624-629), which is atapping test performed with the patient's more affected hand on thekeyboard of a laptop computer. These tests are carried out at baselineand then immediately following each blood sample until patients reachtheir full on-stage, and thereafter at intervals until patients reachtheir baseline off-status. Once patients reach their full on-state,video recordings are performed three times at 20 min intervals. Thefollowing mental and motor tasks, which have been shown to increasedyskinesia (Duriff et al., Mov Disord 1999, 14, 242-245) are monitoredduring each video session: (1) sitting still for 1 minute; (2)performing mental calculations; (3) putting on and buttoning a coat; (4)picking up and drinking from a cup of water; and (5) walking. Videotapesare scored using, for example, versions of the Goetz Rating Scale andthe Abnormal Involuntary Movements Scale to document a possible increasein test compound induced dyskinesia.

Actual occurrence and severity of dyskinesia is measured with aDyskinesia Monitor (Manson et al., J Neurol Neurosurg Psychiatry 2000,68, 196-201). The device is taped to a patient's shoulder on their moreaffected side. The monitor records during the entire time of achallenging session and provides a measure of the frequency and severityof occurring dyskinesias.

Results can be analyzed using appropriate statistical methods.

Example 15: Efficacy of Creatine Prodrugs in MPTP Induced NeurotoxicityAnimal Model of Parkinson's Disease

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin thatproduces a Parkinsonian syndrome in both man and experimental animals.Studies of the mechanism of MPTP neurotoxicity show that it involves thegeneration of a major metabolite, MPP⁺, formed by the activity ofmonoamine oxidase on MPTP. Inhibitors of monoamine oxidase block theneurotoxicity of MPTP in both mice and primates. The specificity of theneurotoxic effects of MPP⁺ for dopaminergic neurons appears to be due tothe uptake of MPP⁺ by the synaptic dopamine transporter. Blockers ofthis transporter prevent MPP⁺ neurotoxicity. MPP⁺ has been shown to be arelatively specific inhibitor of mitochondrial complex I activity,binding to complex I at the retenone binding site and impairingoxidative phosphorylation. In vivo studies have shown that MPTP candeplete striatal ATP concentrations in mice. It has been demonstratedthat MPP⁺ administered intra-striatally in rats produces significantdepletion of ATP as well as increased lactate concentration confined tothe striatum at the site of the injections. Compounds that enhance ATPproduction can protect against MPTP toxicity in mice.

A creatine prodrug is administered to animals such as mice or rats forthree weeks before treatment with MPTP. MPTP is administered at anappropriate dose, dosing interval, and mode of administration for 1 weekbefore sacrifice. Control groups receive either normal saline or MPTPhydrochloride alone. Following sacrifice the two striate are rapidlydissected and placed in chilled 0.1 M perchloric acid. Tissue issubsequently sonicated and aliquots analyzed for protein content using afluorometer assay. Dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), andhomovanillic acid (HVA) are also quantified. Concentrations of dopamineand metabolites are expressed as nmol/mg protein.

Creatine prodrugs that protect against DOPAC depletion induced by MPTP,HVA, and/or dopamine depletion are neuroprotective and therefore can beuseful for the treatment of Parkinson's disease.

Example 16: Evaluation of Potential Anti-Parkinsonian Activity Using aHaloperidol-Induced Hypolocomotion Animal Model

It has been demonstrated that adenosine antagonists, such astheophylline, can reverse the behavioral depressant effects of dopamineantagonists, such as haloperidol, in rodents and is considered a validmethod for screening drugs with potential antiparkinsonian effects(Mandhane, et al., Eur. J. Pharmacol. 1997, 328, 135-141). The abilityof creatine prodrugs to block haloperidol-induced deficits in locomotoractivity in mice can be used to assess both in vivo and potentialanti-Parkinsonian efficacy.

Mice used in the experiments are housed in a controlled environment andallowed to acclimatize before experimental use. 1.5 h before testing,mice are administered 0.2 mg/kg haloperidol, a dose that reducesbaseline locomotor activity by at least 50%. A test compound isadministered 5-60 min prior to testing. The animals are then placedindividually into clean, clear polycarbonate cages with a flatperforated lid. Horizontal locomotor activity is determined by placingthe cages within a frame containing a 3×6 array of photocells interfacedto a computer used to tabulate beam interrupts. Mice are leftundisturbed to explore for 1 h, and the number of beam interruptionsmade during this period serves as an indicator of locomotor activity,which is compared with data for control animals for statisticallysignificant differences.

Example 17: 6-Hydroxydopamine Animal Model of Parkinson's Disease

The neurochemical deficits seen in Parkinson's disease can be reproducedby local injection of the dopaminergic neurotoxin, 6-hydroxydopamine(6-OHDA) into brain regions containing either the cell bodies or axonalfibers of the nigrostriatal neurons. By unilaterally lesioning thenigrostriatal pathway on only one-side of the brain, a behavioralasymmetry in movement inhibition is observed. Althoughunilaterally-lesioned animals are still mobile and capable of selfmaintenance, the remaining dopamine-sensitive neurons on the lesionedside become supersensitive to stimulation. This is demonstrated by theobservation that following systemic administration of dopamine agonists,such as apomorphine, animals show a pronounced rotation in a directioncontralateral to the side of lesioning. The ability of compounds toinduce contralateral rotations in 6-OHDA lesioned rats has been shown tobe a sensitive model to predict drug efficacy in the treatment ofParkinson's disease.

Male Sprague-Dawley rats are housed in a controlled environment andallowed to acclimatize before experimental use. Fifteen minutes prior tosurgery, animals are given an intraperitoneal injection of thenoradrenergic uptake inhibitor desipramine (25 mg/kg) to prevent damageto nondopamine neurons. Animals are then placed in an anaestheticchamber and anaesthetized using a mixture of oxygen and isoflurane. Onceunconscious, the animals are transferred to a stereotaxic frame, whereanesthesia is maintained through a mask. The top of the animal's head isshaved and sterilized using an iodine solution. Once dry, a 2 cm longincision is made along the midline of the scalp and the skin retractedand clipped back to expose the skull. A small hole is then drilledthrough the skull above the injection site. In order to lesion thenigrostriatal pathway, the injection cannula is slowly lowered toposition above the right medial forebrain bundle at −3.2 mm anteriorposterior, -1.5 mm medial lateral from the bregma, and to a depth of 7.2mm below the duramater. Two minutes after lowering the cannula, 6-OHDAis infused at a rate of 0.5 μL/min over 4 min, yielding a final dose of8 μg. The cannula is left in place for an additional 5 min to facilitatediffusion before being slowly withdrawn. The skin is then sutured shut,the animal removed from the stereotaxic frame, and returned to itshousing. The rats are allowed to recover from surgery for two weeksbefore behavioral testing.

Rotational behavior is measured using a rotometer system havingstainless steel bowls (45 cm diameter×0.15 cm high) enclosed in atransparent Plexiglas cover running around the edge of the bowl andextending to a height of 29 cm. To assess rotation, rats are placed in acloth jacket attached to a spring tether connected to an opticalrotometer positioned above the bowl, which assesses movement to the leftor right either as partial (450) or full (3600 rotations).

To reduce stress during administration of a test compound, rats areinitially habituated to the apparatus for 15 min on four consecutivedays. On the test day, rats are given a test compound, e.g., a creatineprodrug. Immediately prior to testing, animals are given a subcutaneousinjection of a subthreshold dose of apomorphine, and then placed in theharness and the number of rotations recorded for one hour. The totalnumber of full contralateral rotations during the hour test periodserves as an index of antiparkinsonian drug efficacy.

Example 18: Animal Studies to Assess the Efficacy of Creatine Prodrugsin Ischemic Injury

Adult male rats are given a creatine prodrug and, after about 24 h, areanesthetized and prepared for coronary artery occlusion. An additionaldose of a creatine prodrug is administered at the start of the procedureand the left main coronary artery occluded for 30 min and then released.The same dose of a creatine prodrug is then administered at appropriateintervals and duration following surgery. The animals are then studiedfor cardiac function. Animals receiving a sham injection (saline)demonstrate a large increase in the left end diastolic pressure,indicative of a dilated, stiff heart secondary to myocardial infarction.Creatine prodrugs that eliminate or reduce the deficit in cardiacfunction compared to sham operated control are useful in preventingischemic injury.

Example 19: Animal Studies to Assess the Ability of Creatine Prodrugs toMaintain Organ Viability

Wistar male rats weighing 300 to 330 g are administered a creatineprodrug or vehicle 24 h prior to removal of the heart for ex vivostudies. Animals are sacrificed with pentobarbital (0.3 mL) andintravenously heparinized (0.2 mL). The hearts are initially allowed toequilibrate for 15 min. The left ventricular balloon is then inflated toa volume that gives an end-diastolic pressure of about 8 mm Hg. A leftventricular pressure-volume curve is constructed by incrementalinflation of the balloon volume by 0.02 mL aliquots. Zero volume isdefined as the point at which the left ventricular end-diastolicpressure is zero. On completion of the pressure-volume curve, the leftventricular balloon is deflated to set end-diastolic pressure back to 8mmHg and the control period is continued for 15 min after check ofcoronary flow. The heart is then arrested with 50 mL Celsior+molecule torest at 4° C. under a pressure of 60 cm H₂O. The heart is then removedand stored for 5 h at 4° C. in a plastic container filled with the samesolution and surrounded with crushed ice.

After storage, the heart is transferred to a Langendorff apparatus. Theballoon catheter is re-inserted into the left ventricle and re-inflatedto the same volume as during the preischemic period. The heart isreperfused for at least 2 h at 37^(o) C. The re-perfusion pressure isset at 50 cm H₂O for 15 min of re-flow and then back to 100 cm H₂O forthe 2 next h. Pacing (320 beats per min) is re-instituted. Isovolumetricmeasurements of contractile indexes and diastolic pressure are taken intriplicate at 25, 45, 60, and 120 min of reperfusion. At this time pointpressure volume curves are obtained and coronary effluent during the 45min reperfusion collected to measure creatine kinase leakage. Improvedleft ventricular pressure following treatment with a prodrug of acreatine analog, as well as improved volume-pressure curve, decrease ofleft diastolic ventricular pressure and decrease of creatine kinaseleakage indicates the ability of the creatine prodrug to maintain organviability.

Example 20: Neuroprotective Effects of Prodrugs of Creatine Analogs in aTransgenic Mouse Model of Huntington's Disease

Transgenic HD mice of the N171-82Q strain and non-transgenic littermatesare treated with a prodrug of a creatine analog or a vehicle from 10weeks of age. The mice are placed on a rotating rod (“rotarod”). Thelength of time at which a mouse falls from the rotarod is recorded as ameasure of motor coordination. The total distance traveled by a mouse isalso recorded as a measure of overall locomotion. Mice administeredcreatine prodrugs that are neuroprotective in the N171-82Q transgenic HDmouse model remain on the rotarod for a longer period of time and travelfurther than mice administered vehicle.

Example 21: Efficacy of Creatine Prodrugs in a Malonate Model ofHuntington's Disease

A series of reversible and irreversible inhibitors of enzymes involvedin energy generating pathways has been used to generate animal modelsfor neurodegenerative diseases such as Parkinson's and Huntington'sdiseases. Inhibitors of succinate dehydrogenase, an enzyme that impactscellular energy homeostasis, has been used to generate a model forHuntington's disease (Brouillet et al., J. Neurochem. 1993, 60, 356-359;Beal et al., J. Neurosci. 1993, 13, 4181-4192; Henshaw et al., BrainResearch 1994, 647, 161-166 (1994); and Beal et al., J. Neurochem. 1993,61, 1147-1150). The enzyme succinate dehydrogenase plays a central rolein both the tricarboxylic acid cycle as well as the electron transportchain in the mitochondria. Malonate is a reversible inhibitor malonateof succinate dehydrogenase. Intrastriatal injections of malonate in ratshave been shown to produce dose dependent striatal excitotoxic lesionsthat are attenuated by both competitive and noncompetitive NMDAantagonists (Henshaw et al., Brain Research 1994, 647, 161-166). Theglutamate release inhibitor, lamotrigine, also attenuates the lesions.Co-injection with succinate blocks the lesions, consistent with aneffect on succinate dehydrogenase. The lesions are accompanied by asignificant reduction in ATP levels as well as significant increase inlactate levels in vivo as shown by chemical shift resonance imaging(Beal et al., J. Neurochem. 1993, 61, 1147-1150). The lesions producedthe same pattern of cellular sparing, which is seen in Huntington'sdisease, supporting malonate challenge as a useful model for theneuropathologic and neurochemical features of Huntington's disease.

To evaluate the effect of creatine prodrugs in this malonate model forHuntington's disease, a creatine prodrug is administered at anappropriate dose, dosing interval, and route, to male Sprague-Dawleyrats. A prodrug is administered for two weeks prior to theadministration of malonate and then for an additional week prior tosacrifice. Malonate is dissolved in distilled deionized water and the pHadjusted to 7.4 with 0.1 M HCl. Intrastriatal injections of 1.5 μL ofmalonate containing 3 μmol are made into the left striatum at the levelof the Bregma 2.4 mm lateral to the midline and 4.5 mm ventral to thedura. Animals are sacrificed at 7 days by decapitation and the brainsquickly removed and placed in ice cold 0.9% saline solution. Brains aresectioned at 2 mm intervals in a brain mold. Slices are then placedposterior side down in 2% 2,3,5-triphenyltetrazolium chloride. Slicesare stained in the dark at room temperature for 30 min and then removedand placed in 4% paraformaldehyde pH 7.3. Lesions, noted by palestaining, are evaluated on the posterior surface of each section. Themeasurements are validated by comparison with measurements obtained onadjacent Nissl stain sections.

Compounds exhibiting a neuroprotective effect and therefore useful intreating Huntington's disease show a reduction in malonate-inducedlesions.

Example 22: Efficacy of Creatine Prodrugs in a Model of CreatineTransporter Disorder

A mouse model of human CrT deficiency has been generated, which enablesdevelopment of treatments for this condition (Skelton et al., PloS One,201, 6 (1), e16187). Mice with exons 2-4 of Slc6a8 flanked by loxP siteswere crossed to Cre:CMV mice to create a line of ubiquitous CrT knockoutexpressing mice. Male CrT−/y (affected) mice lack Cr in the brain andmuscle with significant reductions of Cr in other tissues includingheart and testes. CrT−/y mice show increased path length duringacquisition and reversal learning in the Morris water maze. During probetrials, CrT−/y mice show increased average distance from the platformsite. CrT−/y mice show reduced novel object recognition and conditionedfear memory compared to CrT+/y. CrT−/y mice have increased serotonin and5-hydroxyindole acetic acid in the hippocampus and prefrontal cortex.Ubiquitous CrT knockout mice have learning and memory deficitsresembling human CrT deficiency and this model is useful inunderstanding this disorder and testing creatine prodrugs as therapiesfor this disorder.

To evaluate the effect of creatine prodrugs in the Morris Water Maze(MWM), a creatine prodrug is administered at an appropriate dose, dosinginterval, and route, to male CrT−/y mice. The MWM is a test of spatiallearning and reference memory (Vorhees et al., Nature Protocols 2006, 1:848-858). and animals are tested as described by Skelton et al., BrainRes 2003, 984: 1-10] and Schaefer et al., Neuroscience 2009, 164:1431-1443. Prior to hidden platform testing, visible platform training(cued learning) is conducted for 6 days. During this phase, curtains areclosed around the maze to obscure prominent distal cues and a 10 cmdiameter platform with an orange ball mounted above it on a brass rod isplaced in a predetermined quadrant. On the first day, 6 trials (90 s)are administered with the platform and start in the same position; 2trials per day are given on subsequent days with the start and platformpositions randomized.

The hidden platform portion of the MWM test is conducted in three phases(6 days/phase: acquisition, reversal, and shift) consisting of 4 trialsper day for 6 days for animals to learn the location of the hiddenplatform followed by a single probe trial (no platform) on day 7(Vorhees et al., Nature Protocols 2006, 1: 848-858). Platform diametersare 10 cm for acquisition, 7 cm for reversal (located in the oppositequadrant), and 5 cm for shift, (located in one of the adjacentquadrants). Performance is measured using AnyMaze software (StoeltingCompany, Wood Dale, Ill.). The effect of prodrug treatment is analyzedby comparing performance of control (untreated male CrT−/y mice and/orwild type mice) to prodrug treated mice.

To evaluate the effect of creatine prodrugs in the Conditioned Fearmodel, a creatine prodrug is administered at an appropriate dose, dosinginterval, and route, to male CrT−/y mice. Cued and contextual fear isassessed as described by Peters et al., Science 2010, 328: 1288-1290).On day 1, untreated (control) and treated (prodrug administered) miceare exposed to 30 tones (82 dB, 2 kHz, 30 s on/off cycle) followed by 3tone-footshock pairings (0.5 mA for 1 s). On the following day, animalsare returned to the chamber with no tone or shock presented as a test ofcontextual fear. The next day, animals are placed in the chamber with anovel grid floor. Following 3 min acclimatization, the tone is presentedand freezing behavior scored. Animals are then exposed to 30 cycles of30 s with and 30 s without tone to measure fear extinction. Freezeframesoftware and Coulbourn test chambers are used (Coulbourn Instruments,Allentown, Pa.). Percent time freezing is analyzed. The effect ofprodrug treatment is analyzed by comparing performance of control(untreated male CrT−/y mice and/or wild type mice) to prodrug treatedmice.

To evaluate the effect of creatine prodrugs in the Novel ObjectRecognition (NOR) model, a creatine prodrug is administered at anappropriate dose, dosing interval, and route, to male CrT−/y mice. NORis a test of short-term memory (Clark et al., J Neurosci 2000, 20:8853-8860). Mice are habituated to the arena (91 cm diameter) for 2 days(10 min/day) followed by 2 days (10 min/day) of habituation to twoidentical objects. On the test day, animals are presented with two newidentical objects until 30 s of cumulative observation time is obtained.One hour later memory is tested by presenting the animal with anidentical copy of one of the familiar objects along with a novel object.A discrimination index is calculated by subtracting the time observingthe familiar object from time spent observing the novel object. Theeffect of prodrug treatment is analyzed by comparing performance ofcontrol (untreated male CrT−/y mice and/or wild type mice) to prodrugtreated mice.

Compounds useful in treating creatine transporter disorder will show animprovement in treated male CrT^(−/y) mice relative to untreatedcontrols in one or more of the evaluations outlined above or inalternative models for testing behavior, neurological and/orneuromuscular function.

Example 23: Synthesis of ethyl(N′-hydroxy-N-methylcarbamimidamido)acetate

Ethyl (N′-hydroxy-N-methylcarbamimidamido) acetate can be synthesizedusing the procedure described in Zbinden et al., Bioorganic & MedicinalChemistry Letters, 2005, 15: 5344-5352. Briefly, ethyl[cyano(methyl)amino]acetate, available from MP Biomedicals, Inc., isreflux with hydroxylamine hydrochloride in EtOH to give the titlecompound.

Example 24: Synthesis of tert-butyl2-(3-(tert-butoxycarbonyl)-2-hydroxy-1-methylguanidino)acetate-3-yl)amino]acetate

Step 1: Synthesis of tert-butyl 2-(N-methylcyanamido)acetate

A round bottom flask equipped with a stir bar was charged withtert-butyl 2-(methylamino)acetate (500 mg, 2.75 mmol), potassiumcarbonate (761 mg, 5.50 mmol) and acetonitrile (10 mL). The mixture wasstirred at room temperature for 30 min at which time a solution ofcyanogen bromide (320 mg, 3.025 mmol) in acetonitrile (2 ml) was added.The reaction was allowed to stir overnight at room temperature and thesolvent was decanted leaving behind an insoluble residue. The solventwas then evaporated under reduced pressure and the material was purifiedby flash chromatography eluting with petroleum ether-ethyl acetate(gradient 0% to 40% ethyl acetate) to give tert-butyl2-(N-methylcyanamido)acetate (410 mg, 2.41 mmol, 88% yield) as a whitesolid. ES LC-MS m/z=171 (M+H⁺) & 193 (M+Na⁺).

Step 2: Synthesis of tert-butyl 2-(2-hydroxy-1-methylguanidino)acetate

A round bottom flask equipped with a stir bar was charged withtert-butyl 2-(N-methylcyanamido)acetate (300 mg, 1.76 mmol) andtetrahydrofuran (5 mL). To this mixture was added hydroxylamine (50%aqueous, 583 mg, 8.80 mmol). After 1 hour, 10 mL of water was added andthe mixture was extracted with 5 ml dichloromethane 3 times. The organiclayer was then dried with MgSO4, filtered and concentrated under reducedpressure to give crude tert-butyl 2-(2-hydroxy-1-methylguanidino)acetate(349 mg, 1.72 mmol, 98% yield) as a white solid which was usedimmediately in the next step. ES LC-MS m/z=204 (M+H⁺).

Step 3: Synthesis of tert-butyl2-(3-(tert-butoxycarbonyl)-2-hydroxy-1-methylguanidino)acetate-3-yl)amino]acetate

A round bottom flask equipped with a stir bar was charged withtert-butyl 2-(2-hydroxy-1-methylguanidino)acetate (349 mg, 1.72 mmol)and tetrahydrofuran (5 mL). To this mixture was added di-tert-butyldicarbonate (375 mg, 1.72 mmol). The reaction was stirred at roomtemperature overnight. The solvent was then evaporated under reducedpressure and the material was purified by flash chromatography elutingwith dichloromethane-ethyl acetate (gradient 0% to 30% ethyl acetate) togive tert-butyl2-(3-(tert-butoxycarbonyl)-2-hydroxy-1-methylguanidino)acetate (151 mg,0.50 mmol, 29% yield) as a white solid. ES LC-MS m/z=304 (M+H⁺). ¹H NMR(DIMETHYL SULFOXIDE-d) δ: 5.73 (s, 2H), 3.81 (s, 2H), 2.76 (s, 3H), 1.42(s, 9H), 1.41 (s, 9H).

Example 25: Synthesis of tert-butyl2-(3-(tert-butoxycarbonyl)-2-hydroxy-1-trideuteriomethylguanidino)acetate

Step 1: Synthesis of tert-butyl 2-(4-nitrophenylsulfonamido) acetate

A round bottom flask equipped with a stir bar and under nitrogen wascharged with glycine tert-butyl ester hydrochloride (20 g, 119.76 mmol)and pyridine (260 mL). The mixture was cooled to 0° C. at which time4-nitrobenzenesulfonyl chloride (28.98 g, 131.74 mmol) was addedportion-wise maintaining the mixture below 10° C. The reaction was thenallowed to warm to room temperature. After 18 h at room temperature, thereaction mixture was poured into water (1000 mL). A precipitate formedwhich was filtered and dried under vacuum to give tert-butyl2-(4-nitrophenylsulfonamido) acetate (30.8 g, 97.46 mmol, 81% yield) asa yellow solid. ¹H NMR (CDCl₃) δ: 8.36-8.34 (m, 2H), 8.07-8.05 (m, 2H),5.23 (br s, 1H), 3.75-3.74 (d, J=5.6 Hz, 2H), 1.35 (s, 9H).

Step 2: Synthesis of tert-butyl2-[(4-nitrophenyl)sulfonyl-(trideuteriomethyl)amino]acetate

A round bottom flask equipped with a stir bar and under nitrogen wascharged with tert-butyl 2-(4-nitrophenylsulfonamido) acetate (30.8 g,97.46 mmol), DMF (320 mL) and CD₃I (14.13 g, 97.46 mmol). To thismixture at room temperature was added Cs₂CO₃ (34.85 g, 107.22 mmol) andthe reaction was stirred for 45 min. The reaction mixture was thenpoured into water (1000 mL) and extracted with EtOAc (3×500 mL). Thecombined organic phases were washed with NaCl (500 mL), dried (Na₂SO₄),filtered and the solvent was evaporated under reduced pressure to givetert-butyl 2-[(4-nitrophenyl)sulfonyl-(trideuteriomethyl)amino]acetate(27.8 g, 83.48 mmol, 81% yield as a pale yellow solid. ¹H NMR (CDCl₃) δ:8.36-8.33 (d, J=8.8 Hz, 2H), 8.01-7.99 (d, J=9.2 Hz, 2H), 3.98 (s, 2H),1.38 (s, 9H).

Step 3: Synthesis of tert-butyl2-(tert-butoxycarbonyl(trideuteriomethyl)amino)acetate

A round bottom flask equipped with a stir bar and under nitrogen wascharged tert-butyl2-[(4-nitrophenyl)sulfonyl-(trideuteriomethyl)amino]acetate (27.8 g,83.48 mmol), Cs₂CO₃ (67.83 g, 208.7 mmol) acetonitrile (400 mL) and THF(40 mL). To this solution was added thiophenol (34 mL, 333.93 mmol) andthe reaction was heated at 45° C. for 90 min at which time the reactionmixture was diluted with MTBE (500 mL) and extracted with water (5×100mL). The combined water extracts were washed with MTBE (500 mL) and tothe water mixture was added DCM (500 mL) followed by (BOC)₂O (36.4 g,166.97 mmol). The biphasic reaction mixture was stirred vigorouslyovernight. The phases were then separated and the aqueous layer wasextracted with DCM (500 mL×5). The combined organic phases dried(Na₂SO₄), filtered and the solvent was evaporated under reducedpressure. The material was purified by chromatography using a 120 gsilica cartridge eluting with heptanes—EtOAc (gradient 0% to 30% EtOAc)to give. tert-butyl2-(tert-butoxycarbonyl(trideuteriomethyl)amino)acetate (6 g, 24.19 mmol,28% yield) as a colorless oil. ¹H NMR (CDCl₃) δ: 3.84-3.74 (m, 2H),1.45-1.41 (m, 18H).

Step 4: Synthesis of tert-butyl 2-(N-trideuterio methylamino)acetate TFAsalt

A round bottom flask equipped with a stir bar was charged withtert-butyl 2-(tert-butoxycarbonyl(trideuteriomethyl)amino)acetate (500mg, 2.02 mmol) and dichloromethane (2 mL). The mixture was cooled to 0°C. at which time 1 mL of trifluoroacetic acid (TFA) was added. Themixture was then stirred at 0° C. for 3 hours. The solvent was thenevaporated under reduced pressure to give tert-butyl 2-(N-trideuteriomethylamino)acetate TFA salt (320 mg, 2.02 mmol, 100% yield) as a palebrown oil, which was used directly in the next step. ES LC-MS m/z=149(M+H⁺).

Step 5: Synthesis of tert-butyl 2-(N-trideuteriomethylcyanamido)acetate

A round bottom flask equipped with a stir bar was charged withtert-butyl 2-(N-trideuterio methylamino)acetate TFA salt (320 mg, 2.02mmol), potassium carbonate (837 mg, 6.06 mmol) and acetonitrile (10 mL).The mixture was stirred at room temperature for 0.5 hours, at which timea solution of cyanogen bromide (235 mg, 2.22 mmol) in acetonitrile (2ml) was added. The reaction was allowed to stir overnight at roomtemperature. The solvent was then decanted leaving behind an insolubleresidue. The solvent was evaporated under reduced pressure and thenpurified by flash chromatography eluting with petroleum ether-ethylacetate (gradient 0% to 40% ethyl acetate) to give tert-butyl2-(N-trideuteriomethylcyanamido)acetate (260 mg, 1.50 mmol, 74% yield)as a white solid. ES LC-MS m/z=174 (M+H⁺) & 196 (M+Na⁺).

Step 6: Synthesis of tert-butyl2-(2-hydroxy-1-trideuteriomethylguanidino)acetate

A round bottom flask equipped with a stir bar was charged withtert-butyl 2-(N-trideuteriomethylcyanamido)acetate (260 mg, 1.50 mmol)and tetrahydrofuran (5 mL). To this mixture was added hydroxylamine (50%aqueous, 495 mg, 7.50 mmol). After 1 hour, 10 mL of water was added andthe mixture was extracted with 5 mL of dichloromethane 3 times. Theorganic layer was dried with MgSO4, filtered and concentrated underreduced pressure to give tert-butyl2-(2-hydroxy-1-trideuteriomethylguanidino)acetate (298 mg, 1.45 mmol,97% yield) as a white solid which was used immediately in the next step.ES LC-MS m/z=207 (M+H⁺).

Step 7: Synthesis of tert-butyl2-(3-(tert-butoxycarbonyl)-2-hydroxy-1-trideuteriomethylguanidino)acetate

A round bottom flask equipped with a stir bar was charged withtert-butyl 2-(2-hydroxy-1-trideuteriomethylguanidino)acetate (298 mg,1.45 mmol) and tetrahydrofuran (5 mL). To this mixture was addeddi-tert-butyl dicarbonate (316 mg, 1.72 mmol). The reaction was stirredat room temperature overnight. The solvent was then evaporated underreduced pressure and the material was purified by flash chromatographyeluting with dichloromethane-ethyl acetate (gradient 0% to 30% ethylacetate) to give tert-butyl2-(3-(tert-butoxycarbonyl)-2-hydroxy-1-trideuteriomethylguanidino)acetate(90 mg, 0.29 mmol, 20% yield) as a white solid. ES LC-MS m/z=307 (M+H⁺).¹H NMR (DIMETHYL SULFOXIDE-d) δ: 5.71 (s, 2H), 3.81 (s, 2H), 1.43 (s,9H), 1.41 (s, 9H).

Other creatine prodrugs and derivatives thereof can be synthesized usingthe procedure described above by the selection of the appropriatestarting material.

Example 26: Synthesis of ethyl2-[methyl-(5-oxo-4H-1,2,4-oxadiazol-3-yl)amino]acetate

A round bottom flask equipped with a stir bar and nitrogen inlet wascharged with ethyl 2-[cyano(methyl)amino]acetate (852 mg, 6.0 mmol) andtetrahydrofuran (30 mL). To this mixture was added hydroxylaminehydrochloride (2.1 g, 30.0 mmol) and triethylamine (1.3 mL, 9.0 mmol).After 18 h, carbonyldiimide (5.8 g, 36.0 mmol) was added and thereaction was allowed 1 h. The solvent was decanted leaving behind aninsoluble residue. The solvent was evaporated under reduced pressure andthe material was purified by reverse phase chromatography eluting withwater-acetonitrile modified with 0.1% trifluoroacetic acid. This gaveethyl 2-[methyl-(5-oxo-4H-1,2,4-oxadiazol-3-yl)amino]acetate as a whitesolid: 80 mg, 0.40 mmol, 7% yield. ES LC-MS m/z=202 (M+H⁺). ¹H NMR(CHLOROFORM-d) δ: 4.25 (q, J=7.2 Hz, 2H), 3.96 (s, 2H), 3.04 (s, 3H),1.31 (t, J=7.1 Hz, 3H). Melting point 120-123° C.

Ethyl 2-[methyl-(5-oxo-4H-1,2,4-oxadiazol-3-yl)amino]acetate can also besynthesized using the procedure described in Kitamure et al, Chem.Pharm. Bull., 2001, 49(3) 268-277.

Example 27: Synthesis of2-[methyl-(5-oxo-4H-1,2,4-oxadiazol-3-yl)amino]acetic acid

A round bottom flask equipped with a stir bar and nitrogen inlet wascharged with ethyl2-[methyl-(5-oxo-4H-1,2,4-oxadiazol-3-yl)amino]acetate (20 mg, 0.1mmol), tetrahydrofuran (5 mL) and water (5 mL). To this mixture wasadded lithium hydroxide mono hydrate (4 mg, 0.1 mmol). After 1 hour thesolvent was evaporated under reduced pressure and the material waspurified by reverse phase chromatography eluting with water-acetonitrilemodified with 0.1% trifluoroacetic acid. This gave2-[methyl-(5-oxo-4H-1,2,4-oxadiazol-3-yl)amino]acetic acid as a whitesolid: 15 mg, 0.09 mmol, 90% yield. ES LC-MS m/z=174 (M+H⁺). ¹H NMR(METHANOL-d4) δ: 3.97 (s, 2H), 2.96 (s, 3H). Melting point 150-155° C.

Other creatine prodrugs and derivatives thereof can be synthesized usingthe procedure described above by the selection of the appropriatestarting material.

Example 28: Synthesis of alkyl2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetate

Step 1: Synthesis of methyl2-[ethoxycarbonylcarbamothioyl(trideuteriomethyl)amino]acetate

A round bottom flask equipped with a stir bar and nitrogen inlet wascharged with ethyl N-(thioxomethylene)carbamate (4.1 mL, 35.0 mmol) anddichloromethane (300 mL). The mixture was cooled to 0° C. and methyl2-(trideuteriomethylamino)acetate trifluoroacetic acid salt (7.70 g,35.0 mmol) was added followed by triethylamine (4.9 mL, 35.0 mmol). Themixture was stirred overnight with warming to room temperature. Themixture was washed with 1N HCl (100 mL), dried (Na₂SO₄) and the solventwas evaporated under reduced pressure. The material was purified bychromatography using a 120 g silica cartridge eluting with heptane-ethylacetate, gradient 0 to 30% ethyl acetate. This gave methyl2-[ethoxycarbonylcarbamothioyl(trideuteriomethyl)amino]acetate: 8.0 g,33.8 mmol, 96% yield. ES LC-MS m/z=238 (M+H⁺). ¹H NMR (CHLOROFORM-d) δ:7.38 (br s, 1H), 4.41-4.59 (m, 2H), 4.20 (q, J=7.1 Hz, 2H), 3.80 (s,3H), 1.31 (t, J=7.1 Hz, 3H).

Step 2: Synthesis of methyl2-[[(Z)—N-ethoxycarbonyl-C-methylsulfanyl-carbonimidoyl]-(trideuteriomethyl)amino]acetate

A round bottom flask equipped with a stir and nitrogen inlet was chargedwith [ethoxycarbonylcarbamothioyl(trideuteriomethyl)amino]acetate (7.82μg, 33.0 mmol), methyliodide (4.1 mL, 66.0 mmol) and tetrahydrofuran(200 mL). To this mixture at room temperature was added sodium hydride(60% in oil; 1.32 g, 33.0 mmol). After 1 hour, the material was pouredinto saturated ammonium chloride solution (100 mL), the aqueous phasewas extracted with ethyl acetate (3×100 mL), dried (Na₂SO₄) and thesolvent was evaporated under reduced pressure. The material was purifiedby chromatography using a 120 g silica cartridge eluting withheptane-ethyl acetate, gradient 0 to 40% ethyl acetate. This gave methyl2-[[(Z)—N-ethoxycarbonyl-C-methylsulfanyl-carbonimidoyl]-(trideuteriomethyl)amino]acetate:8.0 g, 32.0 mmol, 97% yield. ES LC-MS m/z=252 (M+H⁺). ¹H NMR(CHLOROFORM-d) δ: 4.30 (s, 2H), 4.16 (q, J=7.1 Hz, 2H), 3.77 (s, 3H),2.42 (s, 3H), 1.30 (t, J=7.1 Hz, 3H).

Step 3: Synthesis of methyl2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetate

A round bottom flask equipped with a stir bar, Vigreux column andnitrogen inlet was charged with methyl2-[[(Z)—N-ethoxycarbonyl-C-methylsulfanyl-carbonimidoyl]-(trideuteriomethyl)amino]acetate(7.53 g, 30.0 mmol) and pyridine (50 mL). To this mixture was addedhydroxylamine hydrochloride (2.09 g, 30.0 mmol) and the mixture washeated at 60° C. for 1 hour. The solvent was evaporated under reducedpressure. Ethyl acetate (100 mL) and water (100 mL) was added. Thephases were separated and the aqueous phase was extracted with ethylacetate (3×100 mL). The combined organic phases were dried (Na₂SO₄) andthe solvent was evaporated under reduced pressure. The material waspurified by chromatography using a 120 g silica cartridge eluting withheptane-ethyl acetate, gradient 0 to 100% ethyl acetate. This gavemethyl2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetate as awhite solid: 3.0 g, 15.8 mmol, 53% yield. ES LC-MS m/z=191 (M+H⁺). ¹HNMR (CHLOROFORM-d) δ: 3.98 (s, 2H), 3.79 (s, 3H). Melting point 120-125°C.

Step 4: Synthesis of2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetic acid

A round bottom flask equipped with a stir bar and nitrogen inlet wascharged with methyl2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetate (95mg, 0.5 mmol), tetrahydrofuran (5 mL) and water (5 mL). To this mixturewas added lithium hydroxide mono hydrate (21 mg, 0.5 mmol). After 1 hourthe solvent was evaporated under reduced pressure and the material waspurified by reverse phase chromatography eluting with water-acetonitrilemodified with 0.1% trifluoroacetic acid. This gave2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetic acidas a white solid: 50 mg, 0.28 mmol, 57% yield. ES LC-MS m/z=177 (M+H⁺).¹H NMR (METHANOL-d4) δ: 3.98 (s, 2H). Melting point 150-155° C.

Step 5A: Synthesis of heptyl2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetate

A scintillation vial equipped with a stir bar was charged with2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetic acid(88 mg, 0.50 mmol), 1-heptanol (58 mg, 0.50 mmol), dimethylaminopyridine (92 mg, 0.75 mmol) and dichloromethane (10 mL). To this mixturewas added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(144 mg, 0.75 mmol). After 1 hour, triethylamine (0.07 mL, 0.50 mmol)was added. The reaction was allowed another 1 hour. The solvent wasevaporated under reduced pressure and the material was purified byreverse phase chromatography eluting with water-acetonitrile modifiedwith 0.1% trifluoroacetic acid. This gave heptyl2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetate as awhite solid: 70 mg, 0.25 mmol, 50% yield. ES LC-MS m/z=275 (M+H⁺). ¹HNMR (CHLOROFORM-d) δ: 11.06 (br s, 1H), 4.17 (t, J=6.8 Hz, 2H), 3.95 (s,2H), 1.56-1.82 (m, 4H), 1.19-1.44 (m, 7H), 0.85-0.94 (m, 2H). Meltingpoint 110-115° C.

Step 5B: Synthesis of ethyl2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetate

A scintillation vial equipped with a stir bar was charged with2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetic acid(176 mg, 1.00 mmol), ethanol (46 mg, 1.00 mmol), dimethylamino pyridine(183 mg, 1.50 mmol) and dichloromethane (20 mL). To this mixture wasadded N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (288mg, 1.50 mmol). After 1 hour, triethylamine (0.14 mL, 1.00 mmol) wasadded. The reaction was allowed another 1 hour. The solvent wasevaporated under reduced pressure and the material was purified byreverse phase chromatography eluting with water-acetonitrile modifiedwith 0.1% trifluoroacetic acid. This gave ethyl2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetate as awhite solid: 133 mg, 0.65 mmol, 65% yield. ES LC-MS m/z=205 (M+H⁺). ¹HNMR (CHLOROFORM-d) δ: 11.13 (br s, 1H), 4.24 (q, J=7.2 Hz, 2H), 3.96 (s,2H), 1.30 (t, J=7.1 Hz, 3H). Melting point 12-130° C.

Step 5C: Synthesis of isopropyl2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetate

A scintillation vial equipped with a stir bar was charged with2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetic acid(176 mg, 1.00 mmol), isopropanol (60 mg, 1.00 mmol), dimethylaminopyridine (183 mg, 1.50 mmol) and dichloromethane (20 mL). To thismixture was added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (288 mg, 1.50 mmol). After 1 hour, triethylamine (0.14 mL,1.00 mmol) was added. The reaction was allowed another 1 hour. Thesolvent was evaporated under reduced pressure and the material waspurified by reverse phase chromatography eluting with water-acetonitrilemodified with 0.1% trifluoroacetic acid. This gave isopropyl2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetate as awhite solid: 139 mg, 0.64 mmol, 64% yield. ES LC-MS m/z=219 (M+H⁺). ¹HNMR (CHLOROFORM-d) δ: 11.09 (br s, 1H), 5.10 (sept, J=6.3 Hz, 1H), 3.91(s, 2H), 1.28 (d, J=6.2 Hz, 6H). Melting point 143-145° C.

Example 29: Synthesis of3-[2-hydroxyethyl(trideuteriomethyl)amino]-2H-1,2,4-oxadiazol-5-one

A round bottom flask equipped with a stir bar and nitrogen inlet wascharged with2-[(5-oxo-2H-1,2,4-oxadiazol-3-yl)-(trideuteriomethyl)amino]acetic acid(528 mg, 3.0 mmol) and tetrahydrofuran (THF) (60 mL). To this mixturewas added BH₃.THF (1.0 M in THF) (6.0 mL, 6.0 mmol). After 2 h, thereaction was quenched with methanol (5 mL) and the solvent wasevaporated under reduced pressure. The material was purified by reversephase chromatography eluting with water-acetonitrile modified with 0.1%trifluoroacetic acid. This gave3-[2-hydroxyethyl(trideuteriomethyl)amino]-2H-1,2,4-oxadiazol-5-one as awhite solid: 372 mg, 2.30 mmol, 77% yield. ES LC-MS m/z=163 (M+H⁺). ¹HNMR (METHANOL-d4) δ: 3.70 (t, J=5.4 Hz, 3H), 3.33 (s, 1H). Melting point98-104° C.

Example 30: Synthesis of 3-imino-4-(methyl-d3)-1,2,4-oxadiazinan-6-oneand 3-amino-4-(methyl-d3)-4,5-dihydro-6H-1,2,4-oxadiazin-6-one

Step 1: Synthesis of methyl 2-[cyano(trideuteriomethyl)amino]acetate

A flask was fitted with methyl 2-(trideuteriomethylamino) acetate (3.00g, 21.04 mmol, HCl salt), K₂CO₃ (5.82 g, 42.08 mmol) and MeOH (20.00mL). Then carbononitridic bromide (2.23 g, 21.04 mmol) was added. Afterthat the reaction mixture was stirred for about 5 hour at 25° C. Thereaction mixture was evaporated to afford the residue. H₂O (20 mL) wasadded and EtOAc (35 mL×5) was used to extract the product. The organiclayer was washed by brine (20 mL), dried over anhydrous Na₂SO₄ andevaporated to afford methyl 2-[cyano(trideuteriomethyl)amino]acetate(2.00 g, 72.48% yield) as yellow red oil. ¹H NMR (MeOD, 400 MHz) δ: 3.91(s, 2H), 3.79 (s, 3H).

Step 2: Synthesis of 3-imino-4-(methyl-d3)-1,2,4-oxadiazinan-6-one and3-amino-4-(methyl-d3)-4,5-dihydro-6H-1,2,4-oxadiazin-6-one

A flask was fitted with methyl 2-[cyano(trideuteriomethyl)amino]acetate(1.00 g, 7.62 mmol), hydroxylamine (2.12 g, 30.50 mmol, HCl salt) andAcONa (2.81 g, 34.31 mmol) in MeOH (20.00 mL). The reaction mixture wasstirred at 25° C. for about 4 h. The reaction solution was evaporated toremove most of MeOH. Then H₂O (about 3 mL) was added to make all thesolid dissolve. Half of the solution was directly purified by specialprep-HPLC (neutral) to afford 4 peaks with desired MS. Peak C, 39.7 mg,light pink. ES LC-MS m/z=130 (M+H⁺). ¹H NMR (DMSO-d6) δ: 3.67 (s, 2H).Peak D, 70.7 mg, white solid. ES LC-MS m/z=130 (M+H⁺). ¹H NMR (DMSO-d6)δ: 3.64 (s, 2H). The remaining peaks may correspond to3-hydroxy-2-imino-1-(methyl-d3)imidazolidin-4-one,(Z)-2-(hydroxyimino)-1-(methyl-d3)imidazolidin-4-one, or(E)-2-(hydroxyimino)-1-(methyl-d3)imidazolidin-4-one.

Example 31: Synthesis of4-amino-2,5-dimethyl-5,6-dihydro-1,3,5-oxadiazepin-7(2H)-one

The title compound,4-amino-2,5-dimethyl-5,6-dihydro-1,3,5-oxadiazepin-7(2H)-one, can besynthesized from the readily available starting materials creatine(available from Sigma Aldrich) and propan-2-one (acetone, also availablefrom Sigma Aldrich). To a stirred solution of creatine (6.55 g; 50 mmol)and acetone (3.70 mL, 50 mmol) in anhydrous dioxane (100 mL) is addedtrifloroacetic acid (TFA; 2.0 mL; 26 mmol) over 10 minutes. The solutionis stirred at room temperature for 10 min, followed by reflux for 1 h,then concentrated in vacuo to yield the crude product. The crude productis dissolved in a minimum amount of anhydrous dioxane followed byintroduction of a stream of anhydrous HCl gas. The resulting precipitateis filtered and washed with dry diethyl ether to yield the HCl salt ofthe title compound.

By using other aldehydes or ketone instead of propan-2-one, other4-amino-5,6-dihydro-1,3,5-oxadiazepin-7(2H)-one compounds can beprepared.

Other creatine prodrugs can be synthesized using the procedure describedabove by the selection of the appropriate starting material.

All publications, including, e.g., non-patent literature, patentapplications, and patents, cited in this specification are incorporatedherein by reference for all purposes. The invention can be embodied inother specific forms without departing from the spirit or essentialcharacteristics thereof. The foregoing embodiments are therefore to beconsidered in all respects illustrative rather than limiting on theinvention described herein. Scope of the invention is thus indicated bythe appended claims rather than by the foregoing description, and allchanges that come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

1.-34. (canceled)
 35. A compound of Formula (VI), or a pharmaceuticallyacceptable salt, solvate, tautomer or stereoisomer thereof, wherein:

R is —CH₃ or —CD₃; R¹⁰ is hydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂alkyl, C₁₋₁₂ heteroalkyl, substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂cycloalkyl, substituted C₃₋₁₂ cycloalkyl, C₄₋₂₀ cycloalkylalkyl,substituted C₄₋₂₀ cycloalkylalkyl, C₄₋₂₀ heterocycloalkylalkyl,substituted C₄₋₂₀ heterocycloalkylalkyl, C₅₋₁₂ aryl, substituted C₅₋₁₂aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂ heteroaryl, C₆₋₂₀ arylalkyl,substituted C₆₋₂₀ arylalkyl, C₆₋₂₀ heteroarylalkyl, substituted C₆₋₂₀heteroarylalkyl, —C(O)R⁵, —C(O)OR⁵, —C(O)(NR³R⁴), —C(R³R⁴)—C(O)OR²²,—C(R³R⁴)—(O)C(O)R²², —C(R³R⁴)—(O)C(O)—OR²²;

R¹¹ and R¹² are each independently hydrogen, —OR¹³ or —C(O)R⁵, with theproviso that both R¹¹ and R¹² cannot be hydrogen; R¹³ is independentlyhydrogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl,substituted C₁₋₁₂ heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂cycloalkyl, C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl,C₄₋₂₀ heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl,C₅₋₁₂ aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl, substituted C₆₋₂₀ heteroarylalkyl —CH(OR⁵), —C(O)R⁵,—C(O)OR⁵ or —C(O)(NR³R⁴); each R³ and R⁴ is independently hydrogen,C₁₋₁₂ alkyl or substituted C₁₋₁₂ alkyl; R⁵ is hydrogen, C₁₋₁₂ alkyl,substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl, substituted C₁₋₁₂heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂ cycloalkyl, C₄₋₂₀cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl, C₄₋₂₀heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl, C₅₋₁₂aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl; R²² is C₁₋₁₂alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂ heteroalkyl, substituted C₁₋₁₂heteroalkyl, C₃₋₁₂ cycloalkyl, substituted C₃₋₁₂ cycloalkyl, C₄₋₂₀cycloalkylalkyl, substituted C₄₋₂₀ cycloalkylalkyl, C₄₋₂₀heterocycloalkylalkyl, substituted C₄₋₂₀ heterocycloalkylalkyl, C₅₋₁₂aryl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, substituted C₅₋₁₂heteroaryl, C₆₋₂₀ arylalkyl, substituted C₆₋₂₀ arylalkyl, C₆₋₂₀heteroarylalkyl or substituted C₆₋₂₀ heteroarylalkyl; R²³ is hydrogen,C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₅₋₁₂ cycloalkyl, substitutedC₅₋₁₂ cycloalkyl, C₅₋₁₂ aryl, and C₅₋₁₂ substituted aryl, —C(O)—OR²² or—C(O)—R²²; and n is an integer from 1 to
 2. 36. The compound of claim35, wherein R¹⁰ is hydrogen, methyl, ethyl, n-propyl, isopropyl,dodecyl, tert-butyl, phenyl or cyclohexyl.
 37. The compound of claim 35,wherein each R⁵, R¹⁰, and R²² is independently C₁₋₆ alkyl, substitutedC₁₋₆ alkyl, C₃₋₇ cycloalkyl, substituted C₃₋₇ cycloalkyl, C₅₋₇ aryl orsubstituted C₅₋₇ aryl.
 38. The compound of claim 35, wherein each R⁵,R¹⁰ and R²² is independently hydrogen, methyl, ethyl, n-propyl,isopropyl, dodecyl, tert-butyl, phenyl or cyclohexyl.
 39. The compoundof claim 35, wherein R¹¹ and R¹² are each independently —OR¹³ or—C(O)R⁵.
 40. The compound of claim 35, wherein the substituted C₁₋₁₂alkyl, substituted C₁₋₁₂ heteroalkyl, substituted C₃₋₁₂ cycloalkyl,substituted C₄₋₂₀ cycloalkylalkyl, substituted C₄₋₂₀heterocycloalkylalkyl, substituted C₅₋₁₂ aryl, substituted C₅₋₁₂heteroaryl, substituted C₆₋₂₀ arylalkyl, substituted C₆₋₂₀heteroarylalkyl, or substituted C₅₋₁₂ cycloalkyl, is C₁₋₁₂ alkyl, C₁₋₁₂heteroalkyl, C₃₋₁₂ cycloalkyl, C₄₋₂₀ cycloalkylalkyl, C₄₋₂₀heterocycloalkylalkyl, substituted C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl, C₆₋₂₀arylalkyl, C₆₋₂₀ heteroarylalkyl, or C₅₋₁₂ cycloalkyl, respectively,substituted with one or more groups selected from halogen, —NO₂, —OH,—NH₂, —CN, —CF₃, —OCF₃, ═O, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₁₋₁₂alkoxy or substituted C₁₋₁₂ alkoxy, —COOR^(10′) wherein R^(10′) ishydrogen, C₁₋₃ alkyl or —(NR^(11′))₂ wherein each R^(11′) isindependently hydrogen or C₁₋₃ alkyl.
 41. The compound of claim 35,wherein the compound of Formula (VI) is a compound of Formula (XXII),Formula (XXIII), Formula (XXIV), Formula (XXV), Formula (XXVI), Formula(XXVII) or Formula (XXVIII) or a pharmaceutically acceptable salt,solvate, tautomer, or stereoisomer thereof; wherein the compound ofFormula (XXII) is:

wherein the compound of Formula (XXIII) is:

wherein the compound of Formula (XXIV) is:

wherein the compound of Formula (XXV) is:

wherein the compound of Formula (XXVI) is:

wherein the compound of Formula (XXVII) is:

wherein the compound of Formula (XXVIII) is:

wherein R is —CH₃ or —CD₃; R^(a) is hydrogen, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl; R³² is hydrogen, methyl,ethyl, n-propyl, isopropyl, tert-butyl, dodecyl, phenyl, cyclohexyl;—CH₂—C(O)OR⁴³, —CH₂—(O)C(O)R⁴³, —CH₂—(O)C(O)OR⁴³ or

R³⁹ is hydrogen, methyl, ethyl, n-propyl, isopropyl, tert-butyl,dodecyl, phenyl or cyclohexyl; each R³³ is independently hydrogen,methyl, ethyl, n-propyl, isopropyl, tert-butyl, dodecyl, phenyl orcyclohexyl; R⁴³ is hydrogen, methyl, ethyl, n-propyl, isopropyl,tert-butyl, dodecyl, phenyl or cyclohexyl; and R³ and R⁴ are eachindependently hydrogen, C₁₋₁₂ alkyl or substituted C₁₋₁₂ alkyl.
 42. Thecompound of claim 35, which is selected from

or a pharmaceutically acceptable salt, solvate, tautomer, orstereoisomer thereof.
 43. A pharmaceutical composition comprising atherapeutically effective amount of at least one compound of claim 35,and a pharmaceutically acceptable vehicle.
 44. The pharmaceuticalcomposition of claim 43, which is in one or more sustained release oraldosage forms.
 45. The pharmaceutical composition of claim 43, whereinthe at least one compound is present in an amount effective for thetreatment of a disease in a patient wherein the disease is ischemia,oxidative stress, a neurodegenerative disease, ischemic reperfusioninjury, a cardiovascular disease, a genetic disease affecting thecreatine kinase system, multiple sclerosis, a psychotic disorder, andmuscle fatigue; an amount sufficient to effect energy homeostasis in atissue or an organ affected by a disease; an amount effective for theenhancement of muscle strength in a patient; an amount effective for theimprovement of the viability of a tissue or an organ; or an amounteffective for the improvement of the viability of cells.
 46. Thepharmaceutical composition of claim 43, wherein the at least onecompound is present in an amount effective for the treatment of agenetic disease affecting the creatine kinase system.
 47. Thepharmaceutical composition of claim 43, wherein the at least onecompound is present in an amount effective for the treatment of acreatine transporter disorder.
 48. The pharmaceutical composition ofclaim 43, wherein the at least one compound is present in an amounteffective for the treatment of a creatine synthesis disorder.
 49. Amethod of delivering creatine to a patient in need thereof comprisingadministering a therapeutically effective amount of the pharmaceuticalcomposition of claim
 43. 50. A method of treating ischemia, oxidativestress, a neurodegenerative disease, ischemic reperfusion injury, acardiovascular disease, a genetic disease affecting the creatine kinasesystem, multiple sclerosis, a psychotic disorder or muscle fatigue in apatient in need thereof comprising administering a therapeuticallyeffective amount of the pharmaceutical composition of claim
 43. 51. Themethod of claim 50, wherein the genetic disease affecting the creatinekinase system is a creatine transporter disorder or a creatine synthesisdisorder.
 52. A method of treating a creatine transporter disorder in apatient in need thereof comprising administering a therapeuticallyeffective amount of the pharmaceutical composition of claim 43.